Antisense modulation of human Rho family gene expression

ABSTRACT

This invention provides compositions and methods for modulating expression of members of the human Rho gene family, which encode low molecular weight GTPases that act as molecular switches in signal transduction. In preferred embodiments, Rho family members include RhoA, RhoB, RhoC, RhoG, Rac1 and cdc42. This invention is also directed to methods for inhibiting hyperproliferation of cells; these methods can be used diagnostically or therapeutically. Furthermore, this invention is directed to treatment of conditions associated with expression of the human Rho family members, particularly in hyperproliferative disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation of U.S. Ser. No. 09/387,341 filed Aug. 31, 1999 which is a continuation-in-part application of U.S. Ser. No. 09/156,424 filed Sep. 18, 1998, now issued as U.S. Pat. No. 5,945,290, and U.S. Ser. No. 09/156,979 filed Sep. 18, 1998, now issued as U.S. Pat. No. 5,962,672, and U.S. Ser. No. 09/156,807 filed Sep. 18, 1998, now issued as U.S. Pat. No. 6,030,786, and U.S. Ser. No. 09/161,015, filed on Sep. 25, 1998, now issued as U.S. Pat. No. 5,965,370.

FIELD OF THE INVENTION

[0002] This invention relates to compositions and methods for modulating expression of members of the human Rho gene family, which encode low molecular weight GTPases that act as molecular switches in signal transduction. This invention is also directed to methods for inhibiting hyperproliferation of cells; these methods can be used diagnostically or therapeutically. Furthermore, this invention is directed to treatment of conditions associated with expression of the human Rho family member genes.

BACKGROUND OF THE INVENTION

[0003] The Rho family of genes are a sub-family of low molecular weight GTPases and are related to each other based on sequence homology and function (Vojtek, A. B., and Cooper, J. A., Cell 1995, 82, 527-529). Other sub-families include Ras, Rab, Arf, and Ran. As GTPases, these proteins bind and hydrolyze GTP. In an active state, they bind to GTP and transduce signals of other proteins in signal transduction pathways. In their inactive state, they are bound to GDP. Members of the Rho family are typically involved in regulation of the actin cytoskeleton. Members of the Rho family include RhoA, RhoB, RhoC, RhoD, RhoE, RhoG, Rac1, Rac2, Rac3 and Cdc42.

[0004] Each class appears to have a unique function in actin reorganization. Rho has been shown to be essential for the formation of stress fibers and focal adhesions (Ridley, A. J. and Hall, A., Cell 1992, 70, 389-399). Focal adhesions are an area of the cell where integrin receptors cluster and extracellular matrix proteins such as fibronectin and collagen are bound. Stress fibers attach at these focal adhesions within a cell. Rac has been shown to be essential for the formation of membrane ruffles, which results from the formation of large vesicles within the cell (Ridley, A. J., et al., Cell 1992, 70, 401-410). Cdc42 (also known as cdc42Hs and G25K) regulates the formation of filopodia, short bundles of actin filaments that protrude from a cell (Nobes, C. D. and Hall, A., Cell 1995, 81, 53-62). Such activities on cell morphology may play an important role in cell motility, cytokinesis, and endocytosis.

[0005] Additional functions for the Rho family have begun to be elucidated. Rac and Rho have been found to promote cadherin-based cell-cell adhesion (Takaishi, K., et al., J. Cell Biol. 1997, 139, 1047-1059). Rac1 and Cdc42 play a critical role in the c-jun amino-terminal kinase (JNK)/stress-activated protein kinase (SAPK) signaling pathway, thereby, potentially having an important role in gene transcription (Coso, O. A. et al., Cell 1995, 81, 1137-1146). RhoA, Rac1 and Cdc42 also regulate transcription through JNK-independent pathways by binding to either serum response factor (SRF; Hill, C. S., et al., Cell 1995, 81, 1159-1170) or NF-κB (Perona, R., et al., Genes and Develop. 1997, 11, 463-475).

[0006] Members of the Rac subfamily have also been found to regulate oxygen radical production. Both Rac1 (Sundaresan, M., et al., Biochem. J. 1996, 318, 379-382) and Rac2 (Knaus, U. G., et al., Science 1991, 254, 1512-1515) are involved in this process.

[0007] Members of the Rho family are thought to be involved in various disease processes, including cancer. Rho, Rac and Cdc42 all play a role in Ras transformation. Rac was found to essential for transformation by Ras, but not RafCAAX, a modified Raf kinase with a localization signal from K-ras (Qiu, R.-G., et al., Nature 1995 374, 457-459). Rho is not essential for Ras transformation, but acts cooperatively in transformation by Ras and RafCAAX (Qiu, R.-G., et al., Proc. Natl. Acad. Sci. USA 1995, 92, 11781-11785). Cdc42 was also found to be essential for Ras transformation, but its role is distinct from that of Rac (Qiu, R.-G., et al., Mol. Cell Biol. 1997, 17, 3449-3458). In addition to transformation, members of the Rho family may also play a role in invasion and metastasis. Michiels, F. et al. (Nature 1995, 375, 338-340) demonstrated that T-lymphoma cells that constitutively expressed Rac1 became invasive. Yoshioka, K. et al. (J. Biol. Chem. 1998, 273, 5146-5154) found that cells stably transfected with RhoA were also invasive. The RhoB gene has been classified as an immediate-early gene, which means that its transcription is rapidly activated upon exposure to certain growth factors or mitogens. The factors shown to activate RhoB transcription include epidermal growth factor (EGF), platelet-derived growth factor (PDGF), genotoxic stress from UV light, alkylating xenobiotics and the retroviral oncogene v-fps. Each of these stimuli triggers DNA synthesis in cultures of high cell density (Engel et al., J. Biol. Chem., 1998, 273, 9921-9926). The response of RhoB to these factors implies a role for RhoB in wound repair and tissue regeneration upon growth factor stimulation and tumorigenesis upon mitogen stimulation.

[0008] The involvement of Rho family proteins in ras-mediated transformation and tumor cell invasion suggests that they could be novel targets for cancer treatment (Ridley, A. J., Int. J. Biochem. Cell Biol. 1997, 29, 1225-1229). In particular, overexpression of the RhoC gene has been associated with pancreatic cancer. Suwa, H. et al. (Br. J. Cancer, 1998, 77, 147-152) looked for a role of RhoA, RhoB and RhoC genes in ductal adenocarcinoma of the pancreas. They found that expression levels of RhoC were higher in tumors than in normal tissue and that metastatic tumors expressed RhoC at higher levels than primary tumors. Rho C expression is also elevated in a megakaryocytic leukemia cell line, CMK. Takada et al., Exp. Hematol., 1996, 24, 524-530. Manifestations of altered RhoB regulation also appear in disease states, including the development of cancer. Cellular transformation and acquisition of the metastatic phenotype are the two main changes normal cells undergo during the progression to cancer. Expression of constitutively activated forms of RhoB have been shown to cause tumorigenic transformation of NIH 3T3 and Rat1 rodent fibroblasts (Khosravi-Far et al., Adv. Cancer Res., 1998, 72, 57-107). RhoB has also been shown to be overexpressed in human breast cancer tissues (Zalcman et al., Oncogene, 1995, 10, 1935-1945). RhoA is also believed to be involved in the development of cancer. Cellular transformation and acquisition of the metastatic phenotype are the two main changes normal cells undergo during the progression to cancer. Recent studies demonstrate that RhoA-regulated pathways can induce both changes in cells. Injecting cells transformed with rhoA genes directly into the bloodstream of mice produced metastasis, or tumor growth, in distant organs (del Peso et al., Oncogene, 1997, 15, 3047-3057).

[0009] It has also been suggested that inhibition of Rac genes may be useful for preventing reoxygenation injury as it occurs when ischemic cells undergo reperfusion (Kim, K.-S., et al., J. Clin. Invest. 1998, 101, 1821-1826). With reoxygenation, reactive oxygen species are presented to the cell, greatly augmenting cell death. Kim, K.-S., et al. showed that adenoviral-mediated transfer of a dominant negative Rac1 could inhibit the formation of reactive oxygen species and protect cells against hypoxia/reoxygenation-induced cell death. They suggest that inhibition of rac1 would be useful, clinically, in treatment in cases where there is the possibility of reperfusion injury.

[0010] Manifestations of altered RhoA regulation also appear in both injury and disease states. It has been proposed that acute central nervous system trauma may contribute to the development of Alzheimer's disease. Findings that show a high concentration of thrombin, a serine-protease in the blood clotting cascade, localized to the plaques of Alzheimer's disease brains support this claim. An excess of thrombin has been shown to stimulate Rho A activity with a concomitant increase in apoptosis (programmed cell death) (Donovan et al., J. Neurosci., 1997, 17, 5316-5326). These studies also imply a role for RhoA in wound repair and clotting disorders.

[0011] Although members of the Rho family have been implicated in various disease processes including cancer and reoxygenation injury, no effective therapy specifically targeting these proteins is available. Antisense oligonucleotides have been used to study the role of some Rho family members in various physiological processes. Dorseuil, O., et al. (J. Biol. Chem. 1992, 267, 20540-20542) used an 16-mer antisense oligonucleotide targeted to the start site of both Rac1 and Rac2 and demonstrated a dose-dependent reduction in superoxide production in whole cells. Brenner, B., et al. (Biochem. Biophys. Res. Commun. 1997, 231, 802-807) used a similar oligonucleotide (a 15-mer targeted to the start site) and showed that inhibition of Rac2 protein expression prevented L-selectin-induced actin polymerization. An 45-mer antisense oligonucleotide targeted to the 3′-UTR has also been used as a probe for rac1 (Didsbury, J., et al., J. Biol. Chem. 1989, 264, 16378-16382).

[0012] Thus, there remains an unmet need for compositions and methods targeting expression of Rho family members, and disease processes associated there-with.

SUMMARY OF THE INVENTION

[0013] The present invention provides oligonucleotides which are targeted to nucleic acids encoding members of the human Rho gene family and are capable of modulating Rho family members expression. The present invention also provides chimeric oligonucleotides targeted to nucleic acids encoding human Rho family members. The oligonucleotides of the invention are believed to be useful both diagnostically and therapeutically, and are believed to be particularly useful in the methods of the present invention.

[0014] The present invention also comprises methods of modulating the expression of human Rho family members using the oligonucleotides of the invention. Methods of inhibiting Rho family members expression are provided; these methods are believed to be useful both therapeutically and diagnostically. These methods are also useful as tools, for example, for detecting and determining the role of Rho family member expression in various cell functions and physiological processes and conditions and for diagnosing conditions associated with expression of Rho family members.

[0015] The present invention also comprises methods for diagnosing and treating cancer and preventing reoxygenation injury. These methods are believed to be useful, for example, in diagnosing Rho family member-associated disease progression. These methods employ the oligonucleotides of the invention. These methods are believed to be useful both therapeutically, including prophylactically, and as clinical research and diagnostic tools.

DETAILED DESCRIPTION OF THE INVENTION

[0016] Members of the Rho family of GTPases are essential for transformation by Ras and play a role in tumor cell invasion. In addition, the Rac subfamily is a regulator of oxygen radical formation. As such, they represent attractive targets for antineoplastic therapy and preventative agents for radical deoxygenation. In particular, modulation of the expression of RhoC may be useful for the treatment of pancreatic carcinomas and modulation of Rac1 may be useful for preventing ischemia/reperfusion injury.

[0017] Antisense oligonucleotides targeting members of the Rho family represent a novel therapeutic approach.

[0018] The present invention employs antisense compounds, particularly oligonucleotides, for use in modulating the function of nucleic acid molecules encoding Rho family members, ultimately modulating the amount of a Rho family member produced. This is accomplished by providing oligonucleotides which specifically hybridize with nucleic acids, preferably mRNA, encoding a Rho family member.

[0019] This relationship between an antisense compound such as an oligonucleotide and its complementary nucleic acid target, to which it hybridizes, is commonly referred to as “antisense”. “Targeting” an oligonucleotide to a chosen nucleic acid target, in the context of this invention, is a multistep process. The process usually begins with identifying a nucleic acid sequence whose function is to be modulated. This may be, as examples, a cellular gene (or mRNA made from the gene) whose expression is associated with a particular disease state, or a foreign nucleic acid from an infectious agent. In the present invention, the targets are nucleic acids encoding Rho family members; in other words, a gene encoding a Rho family member, or mRNA expressed from a Rho family member gene. mRNA which encodes a Rho family member is presently the preferred target. The targeting process also includes determination of a site or sites within the nucleic acid sequence for the antisense interaction to occur such that modulation of gene expression will result.

[0020] In accordance with this invention, persons of ordinary skill in the art will understand that messenger RNA includes not only the information to encode a protein using the three letter genetic code, but also associated ribonucleotides which form a region known to such persons as the 5′-untranslated region, the 3′-untranslated region, the 5′ cap region and intron/exon junction ribonucleotides. Thus, oligonucleotides may be formulated in accordance with this invention which are targeted wholly or in part to these associated ribonucleotides as well as to the informational ribonucleotides. The oligonucleotide may therefore be specifically hybridizable with a transcription initiation site region, a translation initiation codon region, a 5′ cap region, an intron/exon junction, coding sequences, a translation termination codon region or sequences in the 5′- or 3′-untranslated region. Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon.” A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding a Rho family member, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms “start codon region,” “AUG region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. This region is a preferred target region. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon. This region is a preferred target region. The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Other preferred target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA or corresponding nucleotides on the gene. The 5′ cap of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap. The 5′ cap region may also be a preferred target region.

[0021] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a pre-mRNA transcript to yield one or more mature mRNA. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., exon-exon or intron-exon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. Targeting particular exons in alternatively spliced mRNAs may also be preferred. It has also been found that introns can also be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.

[0022] Once the target site or sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired modulation.

[0023] “Hybridization”, in the context of this invention, means hydrogen bonding, also known as Watson-Crick base pairing, between complementary bases, usually on opposite nucleic acid strands or two regions of a nucleic acid strand. Guanine and cytosine are examples of complementary bases which are known to form three hydrogen bonds between them. Adenine and thymine are examples of complementary bases which form two hydrogen bonds between them.

[0024] “Specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide.

[0025] It is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment or, in the case of in vitro assays, under conditions in which the assays are conducted.

[0026] Hybridization of antisense oligonucleotides with mRNA interferes with one or more of the normal functions of mRNA. The functions of mRNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in by the RNA. Binding of specific protein(s) to the RNA may also be interfered with by antisense oligonucleotide hybridization to the RNA.

[0027] The overall effect of interference with mRNA function is modulation of expression of a Rho family member. In the context of this invention “modulation” means either inhibition or stimulation; i.e., either a decrease or increase in expression. This modulation can be measured in ways which are routine in the art, for example by Northern blot assay of mRNA expression, or reverse transcriptase PCR, as taught in the examples of the instant application or by Western blot or ELISA assay of protein expression, or by an immunoprecipitation assay of protein expression. Effects on cell proliferation or tumor cell growth can also be measured, as taught in the examples of the instant application. Inhibition is presently preferred.

[0028] The oligonucleotides of this invention can be used in diagnostics, therapeutics, prophylaxis, and as research reagents and in kits. Since the oligonucleotides of this invention hybridize to nucleic acids encoding a Rho family member, sandwich, colorimetric and other assays can easily be constructed to exploit this fact. Provision of means for detecting hybridization of oligonucleotide with a Rho family member gene or mRNA can routinely be accomplished. Such provision may include enzyme conjugation, radiolabelling or any other suitable detection systems. Kits for detecting the presence or absence of a Rho family member may also be prepared.

[0029] The present invention is also suitable for diagnosing abnormal proliferative states in tissue or other samples from patients suspected of having a hyperproliferative disease such as cancer. The ability of the oligonucleotides of the present invention to inhibit cell proliferation may be employed to diagnose such states. A number of assays may be formulated employing the present invention, which assays will commonly comprise contacting a tissue sample with an oligonucleotide of the invention under conditions selected to permit detection and, usually, quantitation of such inhibition. In the context of this invention, to “contact” tissues or cells with an oligonucleotide or oligonucleotides means to add the oligonucleotide(s), usually in a liquid carrier, to a cell suspension or tissue sample, either in vitro or ex vivo, or to administer the oligonucleotide(s) to cells or tissues within an animal. Similarly, the present invention can be used to distinguish a Rho family member-associated tumor from tumors having other etiologies, or those associated with one rho family member from another, in order that an efficacious treatment regimen can be designed.

[0030] The oligonucleotides of this invention may also be used for research purposes. Thus, the specific hybridization exhibited by the oligonucleotides may be used for assays, purifications, cellular product preparations and in other methodologies which may be appreciated by persons of ordinary skill in the art.

[0031] In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent intersugar (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced binding to target and increased stability in the presence of nucleases.

[0032] The antisense compounds in accordance with this invention preferably comprise from about 5 to about 50 nucleobases. Particularly preferred are antisense oligonucleotides comprising from about 8 to about 30 nucleobases (i.e. from about 8 to about 30 linked nucleosides). As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

[0033] Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0034] Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and borano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.

[0035] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. No. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361 and 5,625,050.

[0036] Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

[0037] Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.

[0038] In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. Further teaching of PNA compounds can be found in Nielsen et al. (Science, 1991, 254, 1497-1500).

[0039] Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

[0040] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O—, S—, or N-alkyl, O-alkyl-O-alkyl, O—, S—, or N-alkenyl, or O—, S—or N-alkynyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)₂ON(CH₃)₂, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)(CH₃)]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′—O—CH₂CH₂OCH₃, also known as 2′—O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in U.S. patent application Ser. No. 09/016,520, filed on Jan. 30, 1998, which is commonly owned with the instant application and the contents of which are herein incorporated by reference.

[0041] Other preferred modifications include 2′-methoxy (2′—O—CH₃), 2′-aminopropoxy (2′—OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′—F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugars structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,0531 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920.

[0042] Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C or m5c), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering 1990, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, those disclosed by Englisch et al. (Angewandte Chemie, International Edition 1991, 30, 613-722), and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications 1993, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications 1993, CRC Press, Boca Raton, pages 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

[0043] Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.: 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941.

[0044] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett. 1994, 4, 1053-1059), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci. 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let. 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res. 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J. 1991, 10, 1111-1118; Kabanov et al., FEBS Lett. 1990, 259, 327-330; Svinarchuk et al., Biochimie 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett. 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res. 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett. 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al. , J. Pharmacol. Exp. Ther. 1996, 277, 923-937).

[0045] Representative U.S. patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

[0046] The present invention also includes oligonucleotides which are chimeric oligonucleotides. “Chimeric” oligonucleotides or “chimeras,” in the context of this invention, are oligonucleotides which contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of antisense inhibition of gene expression. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art. This RNAse H-mediated cleavage of the RNA target is distinct from the use of ribozymes to cleave nucleic acids. Ribozymes are not comprehended by the present invention.

[0047] Examples of chimeric oligonucleotides include but are not limited to “gapmers,” in which three distinct regions are present, normally with a central region flanked by two regions which are chemically equivalent to each other but distinct from the gap. A preferred example of a gapmer is an oligonucleotide in which a central portion (the “gap”) of the oligonucleotide serves as a substrate for RNase H and is preferably composed of 2′-deoxynucleotides, while the flanking portions (the 5′ and 3′ “wings”) are modified to have greater affinity for the target RNA molecule but are unable to support nuclease activity (e.g., 2′-fluoro- or 2′-O-methoxyethyl-substituted). Other chimeras include “wingmers,” also known in the art as “hemimers,” that is, oligonucleotides with two distinct regions. In a preferred example of a wingmer, the 5′ portion of the oligonucleotide serves as a substrate for RNase H and is preferably composed of 2′-deoxynucleotides, whereas the 3′ portion is modified in such a fashion so as to have greater affinity for the target RNA molecule but is unable to support nuclease activity (e.g., 2′-fluoro- or 2′-O-methoxyethyl-substituted), or vice-versa. In one embodiment, the oligonucleotides of the present invention contain a 2′-O-methoxyethyl (2′—O—CH₂CH₂OCH₃) modification on the sugar moiety of at least one nucleotide. This modification has been shown to increase both affinity of the oligonucleotide for its target and nuclease resistance of the oligonucleotide. According to the invention, one, a plurality, or all of the nucleotide subunits of the oligonucleotides of the invention may bear a 2′-O-methoxyethyl (—O—CH₂CH₂OCH₃) modification. Oligonucleotides comprising a plurality of nucleotide subunits having a 2′-O-methoxyethyl modification can have such a modification on any of the nucleotide subunits within the oligonucleotide, and may be chimeric oligonucleotides. Aside from or in addition to 2′-O-methoxyethyl modifications, oligonucleotides containing other modifications which enhance antisense efficacy, potency or target affinity are also preferred. Chimeric oligonucleotides comprising one or more such modifications are presently preferred.

[0048] The oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the talents of the routineer. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and 2′-alkoxy or 2′-alkoxyalkoxy derivatives, including 2′-O-methoxyethyl oligonucleotides (Martin, P., Helv. Chim. Acta 1995, 78, 486-504). It is also well known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products such as biotin, fluorescein, acridine or psoralen-modified amidites and/or CPG (available from Glen Research, Sterling, Va.) to synthesize fluorescently labeled, biotinylated or other conjugated oligonucleotides.

[0049] The antisense compounds of the present invention include bioequivalent compounds, including pharmaceutically acceptable salts and prodrugs. This is intended to encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of the nucleic acids of the invention and prodrugs of such nucleic acids. APharmaceutically acceptable salts@ are physiologically and pharmaceutically acceptable salts of the nucleic acids of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto (see, for example, Berge et al., “Pharmaceutical Salts,” J. of Pharma. Sci. 1977, 66, 1-19).

[0050] For oligonucleotides, examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.

[0051] The oligonucleotides of the invention may additionally or alternatively be prepared to be delivered in a Aprodrug@ form. The term Aprodrug@ indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993.

[0052] For therapeutic or prophylactic treatment, oligonucleotides are administered in accordance with this invention. Oligonucleotide compounds of the invention may be formulated in a pharmaceutical composition, which may include pharmaceutically acceptable carriers, thickeners, diluents, buffers, preservatives, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients and the like in addition to the oligonucleotide. Such compositions and formulations are comprehended by the present invention.

[0053] Pharmaceutical compositions comprising the oligonucleotides of the present invention may include penetration enhancers in order to enhance the alimentary delivery of the oligonucleotides. Penetration enhancers may be classified as belonging to one of five broad categories, i.e., fatty acids, bile salts, chelating agents, surfactants and non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, 8, 91-192; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems 1990, 7, 1-33). One or more penetration enhancers from one or more of these broad categories may be included.

[0054] Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, recinleate, monoolein (a.k.a. 1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, mono- and di-glycerides and physiologically acceptable salts thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems 1990, 7, 1; El-Hariri et al., J. Pharm. Pharmacol. 1992 44, 651-654).

[0055] The physiological roles of bile include the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996, pages 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus, the term “bile salt” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives.

[0056] Complex formulations comprising one or more penetration enhancers may be used. For example, bile salts may be used in combination with fatty acids to make complex formulations.

[0057] Chelating agents include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)[Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems 1990, 7, 1-33; Buur et al., J. Control Rel. 1990, 14, 43-51). Chelating agents have the added advantage of also serving as DNase inhibitors.

[0058] Surfactants include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page 92); and perfluorochemical emulsions, such as FC-43 (Takahashi et al., J. Pharm. Phamacol. 1988, 40, 252-257).

[0059] Non-surfactants include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol. 1987, 39, 621-626).

[0060] As used herein, “carrier compound” refers to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor.

[0061] In contrast to a carrier compound, a “pharmaceutically acceptable carrier” (excipient) is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The pharmaceutically acceptable carrier may be liquid or solid and is selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutically acceptable carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinyl-pyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrates (e.g., starch, sodium starch glycolate, etc.); or wetting agents (e.g., sodium lauryl sulphate, etc.). Sustained release oral delivery systems and/or enteric coatings for orally administered dosage forms are described in U.S. Pat. No. 4,704,295; 4,556,552; 4,309,406; and 4,309,404.

[0062] The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional compatible pharmaceutically-active materials such as, e.g., antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the composition of present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the invention.

[0063] Regardless of the method by which the oligonucleotides of the invention are introduced into a patient, colloidal dispersion systems may be used as delivery vehicles to enhance the in vivo stability of the oligonucleotides and/or to target the oligonucleotides to a particular organ, tissue or cell type. Colloidal dispersion systems include, but are not limited to, macromolecule complexes, nanocapsules, microspheres, beads and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, liposomes and lipid:oligonucleotide complexes of uncharacterized structure. A preferred colloidal dispersion system is a plurality of liposomes. Liposomes are microscopic spheres having an aqueous core surrounded by one or more outer layers made up of lipids arranged in a bilayer configuration (see, generally, Chonn et al., Current Op. Biotech. 1995, 6, 698-708).

[0064] The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, epidermal, and transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration, e.g., by inhalation or insufflation, or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration.

[0065] Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

[0066] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

[0067] Compositions for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. In some cases it may be more effective to treat a patient with an oligonucleotide of the invention in conjunction with other traditional therapeutic modalities in order to increase the efficacy of a treatment regimen. In the context of the invention, the term “treatment regimen” is meant to encompass therapeutic, palliative and prophylactic modalities. For example, a patient may be treated with conventional chemotherapeutic agents, particularly those used for tumor and cancer treatment. Examples of such chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide, trimetrexate, teniposide, cisplatin and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. Preferred are chemotherapeutic agents which are direct or indirect inhibitors of a Rho family member. These include MTX, Tomudex and fluorinated pyrimidines such as 5-FU and 5-FUdR. When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide).

[0068] The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅₀s found to be effective in vitro and in in vivo animal models. In general, dosage is from 0.01 μg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight, once or more daily, to once every 20 years.

[0069] Thus, in the context of this invention, by “therapeutically effective amount” is meant the amount of the compound which is required to have a therapeutic effect on the treated individual. This amount, which will be apparent to the skilled artisan, will depend upon the age and weight of the individual, the type of disease to be treated, perhaps even the gender of the individual, and other factors which are routinely taken into consideration when designing a drug treatment. A therapeutic effect is assessed in the individual by measuring the effect of the compound on the disease state in the animal. For example, if the disease to be treated is cancer, therapeutic effects are assessed by measuring the rate of growth or the size of the tumor, or by measuring the production of compounds such as cytokines, production of which is an indication of the progress or regression of the tumor.

[0070] The following examples illustrate the present invention and are not intended to limit the same.

EXAMPLES Example 1 Synthesis of Oligonucleotides

[0071] Unmodified oligodeoxynucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine. β-cyanoethyldiisopropyl-phosphoramidites are purchased from Applied Biosystems (Foster City, Calif.). For phosphorothioate oligonucleotides, the standard oxidation bottle was replaced by a 0.2 M solution of ³H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages. The thiation cycle wait step was increased to 68 seconds and was followed by the capping step.

[0072] 2′-methoxy oligonucleotides were synthesized using 2′-methoxyβ-cyanoethyldiisopropyl-phosphoramidites (Chemgenes, Needham, Mass.) and the standard cycle for unmodified oligonucleotides, except the wait step after pulse delivery of tetrazole and base was increased to 360 seconds. Other 2′-alkoxy oligonucleotides were synthesized by a modification of this method, using appropriate 2′-modified amidites such as those available from Glen Research, Inc., Sterling, Va.

[0073] 2′-fluoro oligonucleotides were synthesized as described in Kawasaki et al. (J. Med. Chem. 1993, 36, 831-841). Briefly, the protected nucleoside N⁶-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesized utilizing commercially available 9-β-D-arabinofuranosyladenine as starting material and by modifying literature procedures whereby the 2′-α-fluoro atom is introduced by a S_(N)2-displacement of a 2′-β-O-trifyl group. Thus N⁶-benzoyl-9-β-D-arabinofuranosyladenine was selectively protected in moderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THP and N⁶-benzoyl groups was accomplished using standard methodologies and standard methods were used to obtain the 5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.

[0074] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished using tetraisopropyldisiloxanyl (TPDS) protected 9-β-D-arabinofuranosylguanine as starting material, and conversion to the intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection of the TPDS group was followed by protection of the hydroxyl group with THP to give diisobutyryl di-THP protected arabinofuranosylguanine. Selective O-deacylation and triflation was followed by treatment of the crude product with fluoride, then deprotection of the THP groups. Standard methodologies were used to obtain the 5′-DMT- and 5′-DMT-3′-phosphoramidites.

[0075] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by the modification of a known procedure in which 2,2′-anhydro-1-β-D-arabinofuranosyluracil was treated with 70% hydrogen fluoride-pyridine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′ phosphoramidites.

[0076] 2′-deoxy-2′-fluorocytidine was synthesized via amination of 2′-deoxy-2′-fluorouridine, followed by selective protection to give N⁴-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′ phosphoramidites.

[0077] 2′-(2-methoxyethyl)-modified amidites are synthesized according to Martin, P. (Helv. Chim. Acta 1995, 78, 486-506). For ease of synthesis, the last nucleotide was a deoxynucleotide. 2′—O—CH₂CH₂OCH_(3—) cytosines may be 5-methyl cytosines.

[0078] Synthesis of 5-Methyl Cytosine Monomers

[0079] 2,2′-Anhydro[1-(β-D-arabinofuranosyl)-5-methyluridine]:

[0080] 5-Methyluridine (ribosylthymine, commercially available through Yamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). The mixture was heated to reflux, with stirring, allowing the evolved carbon dioxide gas to be released in a controlled manner. After 1 hour, the slightly darkened solution was concentrated under reduced pressure. The resulting syrup was poured into diethylether (2.5 L), with stirring. The product formed a gum. The ether was decanted and the residue was dissolved in a minimum amount of methanol (ca. 400 mL). The solution was poured into fresh ether (2.5 L) to yield a stiff gum. The ether was decanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for 24 h) to give a solid which was crushed to a light tan powder (57 g, 85% crude yield). The material was used as is for further reactions.

[0081] 2′-O-Methoxyethyl-5-methyluridine:

[0082] 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 L stainless steel pressure vessel and placed in a pre-heated oil bath at 160° C. After heating for 48 hours at 155-160° C., the vessel was opened and the solution evaporated to dryness and triturated with MeOH (200 mL). The residue was suspended in hot acetone (1 L). The insoluble salts were filtered, washed with acetone (150 mL) and the filtrate evaporated. The residue (280 g) was dissolved in CH₃CN (600 mL) and evaporated. A silica gel column (3 kg) was packed in CH₂Cl₂/acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue was dissolved in CH₂Cl₂ (250 mL) and adsorbed onto silica (150 g) prior to loading onto the column. The product was eluted with the packing solvent to give 160 g (63%) of product.

[0083] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine:

[0084] 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporated with pyridine (250 mL) and the dried residue dissolved in pyridine (1.3 L). A first aliquot of dimethoxy-trityl chloride (94.3 g, 0.278 M) was added and the mixture stirred at room temperature for one hour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the reaction stirred for an additional one hour. Methanol (170 mL) was then added to stop the reaction. HPLC showed the presence of approximately 70% product. The solvent was evaporated and triturated with CH₃CN (200 mL). The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500 mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phase was dried over Na₂SO₄, filtered and evaporated. 275 g of residue was obtained. The residue was purified on a 3.5 kg silica gel column, packed and eluted with EtOAc/-Hexane/Acetone (5:5:1) containing 0.5% Et₃NH. The pure fractions were evaporated to give 164 g of product. Approximately 20 g additional was obtained from the impure fractions to give a total yield of 183 g (57%).

[0085] 3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine:

[0086] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) were combined and stirred at room temperature for 24 hours. The reaction was monitored by tlc by first quenching the tlc sample with the addition of MeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL) was added and the mixture evaporated at 35° C. The residue was dissolved in CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodium bicarbonate and 2×200 mL of saturated NaCl. The water layers were back extracted with 200 mL of CHCl₃. The combined organics were dried with sodium sulfate and evaporated to give 122 g of residue (approx. 90% product). The residue was purified on a 3.5 kg silica gel column and eluted using EtOAc/Hexane(4:1). Pure product fractions were evaporated to yield 96 g (84%).

[0087] 3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine:

[0088] A first solution was prepared by dissolving 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L), cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃ was added dropwise, over a 30 minute period, to the stirred solution maintained at 0-10° C., and the resulting mixture stirred for an additional 2 hours. The first solution was added dropwise, over a 45 minute period, to the later solution. The resulting reaction mixture was stored overnight in a cold room. Salts were filtered from the reaction mixture and the solution was evaporated. The residue was dissolved in EtOAc (1 L) and the insoluble solids were removed by filtration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mL of saturated NaCl, dried over sodium sulfate and evaporated. The residue was triturated with EtOAc to give the title compound.

[0089] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine:

[0090] A solution of 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred at room temperature for 2 hours. The dioxane solution was evaporated and the residue azeotroped with MeOH (2×200 mL). The residue was dissolved in MeOH (300 mL) and transferred to a 2 liter stainless steel pressure vessel. MeOH (400 mL) saturated with NH₃ gas was added and the vessel heated to 100° C. for 2 hours (tlc showed complete conversion). The vessel contents were evaporated to dryness and the residue was dissolved in EtOAc (500 mL) and washed once with saturated NaCl (200 mL). The organics were dried over sodium sulfate and the solvent was evaporated to give 85 g (95%) of the title compound.

[0091] N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine:

[0092] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) was added with stirring. After stirring for 3 hours, tlc showed the reaction to be approximately 95% complete. The solvent was evaporated and the residue azeotroped with MEOH (200 mL). The residue was dissolved in CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give a residue (96 g). The residue was chromatographed on a 1.5 kg silica column using EtOAc/Hexane (1:1) containing 0.5% Et₃NH as the eluting solvent. The pure product fractions were evaporated to give 90 g (90%) of the title compound.

[0093] N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite:

[0094] N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74 g, 0.10 M) was dissolved in CH₂Cl₂ (1 L) Tetrazole diisopropylamine (7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M) were added with stirring, under a nitrogen atmosphere. The resulting mixture was stirred for 20 hours at room temperature (tlc showed the reaction to be 95% complete). The reaction mixture was extracted with saturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes were back-extracted with CH₂Cl₂ (300 mL), and the extracts were combined, dried over MgSO₄ and concentrated. The residue obtained was chromatographed on a 1.5 kg silica column using EtOAc\Hexane (3:1) as the eluting solvent. The pure fractions were combined to give 90.6 g (87%) of the title compound.

[0095] 5-methyl-2′-deoxycytidine (5-me-C) containing oligonucleotides were synthesized according to published methods (Sanghvi et al., Nucl. Acids Res. 1993, 21, 3197-3203) using commercially available phosphoramidites (Glen Research, Sterling Va. or ChemGenes, Needham Mass.).

[0096] Oligonucleotides having methylene (methylimino) (MMI) backbones are synthesized according to U.S. Pat. No. 5,378,825, which is coassigned to the assignee of the present invention and is incorporated herein in its entirety. For ease of synthesis, various nucleoside dimers containing MMI linkages were synthesized and incorporated into oligonucleotides. Other nitrogen-containing backbones are synthesized according to WO 92/20823 which is also coassigned to the assignee of the present invention and incorporated herein in its entirety.

[0097] Oligonucleotides having amide backbones are synthesized according to De Mesmaeker et al. (Acc. Chem. Res. 1995, 28, 366-374). The amide moiety is readily accessible by simple and well-known synthetic methods and is compatible with the conditions required for solid phase synthesis of oligonucleotides.

[0098] Oligonucleotides with morpholino backbones are synthesized according to U.S. Pat. No. 5,034,506 (Summerton and Weller).

[0099] Peptide-nucleic acid (PNA) oligomers are synthesized according to P. E. Nielsen et al. (Science 1991, 254, 1497-1500).

[0100] After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55° C. for 18 hours, the oligonucleotides are purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotides were analyzed by polyacrylamide gel electrophoresis on denaturing gels and judged to be at least 85% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in synthesis were periodically checked by ³¹P nuclear magnetic resonance spectroscopy, and for some studies oligonucleotides were purified by HPLC, as described by Chiang et al. (J. Biol. Chem. 1991, 266, 18162). Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Example 2 Human RhoA Oligonucleotide Sequences

[0101] Antisense oligonucleotides were designed to target human RhoA. Target sequence data are from the RhoA cDNA sequence published by Yeramian, P., et al. (Nucleic Acids Res. 1987, 15, 1869); Genbank accession number X05026, provided herein as SEQ ID NO: 1. Oligonucleotides were synthesized primarily with phosphorothioate linkages. Oligonucleotide sequences are shown in Table 1.

[0102] A549 cells, human lung carcinoma cells (obtained from American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (DMEM) low glucose, 10% fetal calf serum, and penicillin (50 units/ml)/streptomycin (50 mg/ml). Cells were passaged at 90-95% confluency. All culture reagents were obtained from Life Technologies (GIBCO BRL, Rockville, Md).

[0103] A549 cells were plated at a starting cell number of approximately 2×10⁵ cells per well. After twenty-four hours, at 80-90% confluency, the cells were washed twice with Opti-Mem (GIBCO BRL) and the oligonucleotide formulated in LIPOFECTIN (GIBCO BRL), a 1:1 (w/w) liposome formulation of the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA), and dioleoyl phosphotidylethanolamine (DOPE) in membrane filtered water, at a constant ratio of 2.5 mg/ml LIPOFECTIN to 100 nM oligonucleotide, in Opti-Mem. For an initial screen, the oligonucleotide concentration was 300 nM. Treatment was for four hours. After treatment, the media was removed and the cells were further incubated in DMEM containing 10% FCS, and penicillin/streptomycin for 24 or 48 hours.

[0104] mRNA was isolated using the MICRO-FASTTRACK kit (Invitrogen, Carlsbad, Calif.), separated on a 1% agarose gel, transferred to Hybond-N+ membrane (Amersham, Arlington Heights, Ill.), a positively charged nylon membrane, and probed. A RhoA probe was generated using asymmetric PCR, in the presence of a[³²P]-dCTP (Amersham), with the following primers: Forward: 5′-TGCAAGCACAGCCCTTATG-3′ SEQ ID NO. 2 Reverse: 5′-TGTCAAAAGGACCCTGGTG-3′ SEQ ID NO. 3

[0105] A glyceraldehyde 3-phosphate dehydrogenase (G3PDH) probe was purchased from Clontech (Palo Alto, Calif.), Catalog Number 9805-1. The probe was labeled by random primer using the Large Fragment of DNA polymerase (Klenow fragment) (GIBCO BRL) as described in Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, 1989. mRNA was quantitated by a PhosphoImager (Molecular Dynamics, Sunnyvale, Calif.). TABLE 1 Nucleotide Sequences of RhoA Oligonucleotides TARGET GENE SEQ NUCLEOTIDE GENE ISIS NUCLEOTIDE SEQUENCE ID CO- TARGET NO. (5′ -> 3′) NO: ORDINATES¹ REGION 16191 AGTCGCAAACTCGGAGAC 4 0085-0102 5′-UTR 16192 TTGCTCAGGCAACGAATC 5 0142-0159 AUG 16193 CTGAACACTATCACCAAGCATG 6 0214-0235 Coding 16194 CTCATCATTCCGAAGATCC 7 0515-0533 Coding 16195 CCAATCCTGTTTGCCATATCTC 8 0592-0613 Coding 16196 CCATCTTTGGTCTTTGCTGAAC 9 0634-0655 Coding 16197 CCAGAGCAGCTCTCGTAGCCA 10 0676-0696 Coding 16198 TCACAAGACAAGGCAACCAG 11 0721-0740 Stop 16199 AGGCCAGTAATCATACACTA 12 0799-0818 3′-UTR 16200 GTTGGCTTCTAAATACTGCT 13 0871-0890 3′-UTR 16201 GGCTGTTAGAGCAGTGTCAA 14 0937-0956 3′-UTR 16202 AGCGCCTGGTGTGTCAGGTG 15 0971-0990 3′-UTR 16203 TAGTTACAGCCTAATTGACA 16 1051-1073 3′-UTR 16913 GGCACCTGTTGGGTGAGCTG 17 16202 control 16914 ACACTCTTGCTTACCGTACCTT 18 16195 control 16915 TCCCGTAAGTGCGGTATCAA 19 16201 control

[0106] Results are shown in Table 2. Oligonucleotides 16193 (SEQ. ID NO. 6), 16195 (SEQ ID NO. 8), 16196 (SEQ ID NO. 9), 16197 (SEQ ID NO. 10), 16198 (SEQ ID NO. 11), 16199 (SEQ ID NO. 12), 16200 (SEQ ID NO. 13), 16201 (SEQ ID NO. 14), and 16202 (SEQ ID NO. 15) gave better than 50% inhibition of RhoA expression. Oligonucleotides 16195 (SEQ ID NO. 8), 16197 (SEQ ID NO. 10), 16199 (SEQ ID NO. 12), 16201 (SEQ ID NO. 14), and 16202 (SEQ ID NO. 15) gave better than 75% inhibition of RhoA expression. TABLE 2 Activities of Phosphorothioate Oligonucleotides Targeted to Human RhoA SEQ GENE ISIS ID TARGET % mRNA % mRNA No: NO: REGION EXPRESSION INHIBITION LIPOFECTIN — — 100.0% 0.0% only 16191 4 5′-UTR 66.4% 33.6% 16192 5 AUCG 68.0% 32.0% 16193 6 Coding 31.9% 68.1% 16194 7 Coding 79.9% 20.1% 16195 8 Coding 3.9% 96.1% 16196 9 Coding 31.4% 68.6% 16197 10 Coding 19.2% 81.8% 16198 11 Stop 46.4% 53.6% 16199 12 3′-UTR 22.9% 77.1% 16200 13 3′-UTR 36.9% 63.1% 16201 14 3′-UTR 22.0% 78.0% 16202 15 3′-UTR 14.4% 85.6% 16203 16 3′-UTR 88.0% 12.0%

Example 3 Dose Response and Specificity of Antisense Oligonucleotide Effects on Human RhoA mRNA Levels in A549 Cells

[0107] Three of the most active oligonucleotides from the initial screen were chosen for dose response assays. These include oligonucleotides 16195 (SEQ ID NO. 8), 16201 (SEQ ID NO. 14), and 16202 (SEQ ID NO. 15). A549 cells were grown, treated and processed as described in Example 2. LIPOFECTIN was added at a ratio of 2.5 mg/ml per 100 nM of oligonucleotide. The control included LIPOFECTIN at a concentration of 7.5 mg/ml. Results are shown in Table 3. Each oligonucleotide showed a dose response effect with maximal inhibition greater than 90%.

[0108] The specificity of these oligonucleotides was investigated using scrambled controls, i.e. oligonucleotides with the same base composition and a scrambled sequence. Oligonucleotide 16915 (SEQ ID NO. 19) is a scrambled control for 16201 (SEQ ID NO. 14) and oligonucleotide 16913 (SEQ ID NO. 17) is a scrambled control for 16202 (SEQ ID NO. 15). Both antisense oligonucleotides showed a dose dependent effect on mRNA expression, while scrambled controls showed much less inhibition which was only seen at higher does. TABLE 3 Dose Response of A549 Cells to RhoA Antisense Oligonucleotides (ASOs) SEQ ID ASO Gene % mRNA % mRNA ISIS # NO: Target Dose Expression Inhibition control — LIPOFECTIN — 100.0% 0.0% only 16195 8 Coding  75 nM 72.7% 27.3% 16195 8 ″ 150 nM 35.0% 65.0% 16195 8 ″ 300 nM 20.3% 79.7% 16201 14 3′-UTR  75 nM 79.1% 20.9% 16201 14 ″ 150 nM 35.7% 64.3% 16201 14 ″ 300 nM 9.5% 90.5% 16202 15 3′-UTR  75 nM 68.7% 31.3% 16202 15 ″ 150 nM 28.8% 71.2% 16202 15 ″ 300 nM 6.1% 93.7%

[0109] TABLE 4 Specificity of RhoA Antisense Oligonucleotides (ASOs) in A549 Cells SEQ ID ASO Gene % mRNA % mRNA ISIS # NO: Target Dose Expression Inhibition control — LIPOFECTIN —  100%   0% only 16201 14 3′-UTR  75 nM 64.4% 35.6% 16201 14 ″ 150 nM 35.3% 64.7% 16201 14 ″ 300 nM  5.7% 94.3% 16915 19 control  75 nM 89.9% 10.1% 16915 19 ″ 150 nM 98.3%  1.7% 16915 19 ″ 300 nM 84.8% 15.2% 16202 15 3′-UTR  75 nM 39.9% 60.1% 16202 15 ″ 150 nM 20.2% 79.8% 16202 15 ″ 300 nM 10.8% 89.2% 16913 17 control  75 nM 97.6%  2.4% 16913 17 ″ 150 nM 89.8% 10.2% 16913 17 ″ 300 nM 55.6% 44.4%

Example 4 Design and Testing of Chimeric (Deoxy Gapped) 2′-O-methoxyethyl RhoA Antisense Oligonucleotides on RhoA Levels in A549 Cells

[0110] Oligonucleotides having SEQ ID NO: 14 were synthesized as a uniformly phosphorothioate or mixed phosphorothioate/phosphodiester chimeric oligonucleotides having variable regions of 2′-O-methoxyethyl (2′-MOE) nucleotides and deoxynucleotides. All 2′-MOE cytosines were 5-methyl-cytosines. Additionally, some oligonucleotides were synthesized with deoxycytosines as 5-methyl-cytosines. Additional oligonucleotides were synthesized, with similar chemistries, as scrambled controls. TABLE 5 Nucleotide Sequences of 16201 Analogues SEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET NO. (5′ -> 3′)¹ NO: CO-ORDINATES² REGION 17130 GsGcCsTsGsTsTsAsGsAsGsCsAsGsTs GsTsCsAsA 14 0937-0956 3′-UTR 17131 GsGsCsTsGsTsTsAsGsAsGsCsAsGsTsGsTsCsAsA 14 0937-0956 3′-UTR 17132 GsGsCsTsGsTsTsAsGsAsGsCsAsGsTsGsTsCsAsA 14 0937-0956 3′-UTR 17133 GsGsCsTsGsTsTsAsGsAsGsCsAsGsTsGsTsCsAsA 14 0937-0956 3′-UTR 17134 GsGsCsTsGsTsTsAsGsAsGsCsAsGsTsGsTsCsAsA 14 0937-0956 3′-UTR 17818 GoGoCsTsGsTsTsAsGsAsGsCsAoGoToGoToCoAoA 14 0937-0956 all 5-meC 17819 ToGoCsGsCsTsAsAsGsTsGsCsGoGoToAoToCoAoA 19 16201 control all 5-meC 18550 TsGsCsGsGsTsAsAsCsTsGsCsGsGsTsAsTsCsAsA 19 16201 control 20459 GsGsCsTsCsTsTsAsGsAsGsCsAsGsTsGsTsCsAsA 14 0937-0956 all 5-meC 21919 GsTsCsGsTsTsAsCsTsCsGsGsAsAsAsTsGsGsAsGsGsC 20 16201 control 21920 AsGsCsTsTsGsTsTsGsAsAsCsGsAsGsTsGsTsCsGsA 21 16201 control 21921 TsGsCsAsGsTsTsCsGsCsAsGsAsGsTsCsTsGsAsA 22 16201 control

[0111] Dose response experiments were performed using chimeric oligonucleotides as discussed in Example 3. Results are shown in Table 6. The introduction of 2′-MOE nucleotides into the sequence improved the maximum inhibition from 60%, with a phosphorothioate oligodeoxynucleotide, to greater than 75%. The exception was the fully modified 2-MOE oligonucleotide which was less effective than the oligodeoxynucleotide. TABLE 6 Dose Response of A549 Cells to RhoA Antisense Gapmer Oliqonucleotides (ASOs) SEQ ID ASO Gene % mRNA % mRNA ISIS # NO: Target Dose Expression Inhibition control — LIPOFECTIN —  100%   0% only 16201 14 3′-UTR  75 nM 119.5%  — 16201 14 ″ 150 nM 54.5% 45.5% 16201 14 ″ 300 nM 39.5% 60.5% 17130 14 3′-UTR  75 nM 56.2% 43.8% 17130 14 ″ 150 nM 31.5% 68.5% 17130 14 ″ 300 nM 14.1% 85.9% 17131 14 3′-UTR  75 nM 55.5% 44.5% 17131 14 ″ 150 nM 35.4% 64.6% 17131 14 ″ 300 nM 24.7% 75.3% 17132 14 3′-UTR  75 nM 71.3% 28.7% 17132 14 ″ 150 nM 31.3% 68.7% 17132 14 ″ 300 nM 13.1% 86.9% 17133 14 3′-UTR  75 nM 41.7% 58.3% 17133 14 ″ 150 nM 33.8% 66.2% 17133 14 ″ 300 nM 14.4% 85.6% 17134 14 3′-UTR  75 nM 76.6% 23.4% 17134 14 ″ 150 nM 35.9% 64.1% 17134 14 ″ 300 nM 68.5% 31.5%

Example 5 Time Course of Antisense Oligonucleotide Effects on Human RhoA Protein Levels in A549 Cells

[0112] Oligonucleotide 17131 was tested by treating for varying times and measuring the effect of the oligo on RhoA protein levels. A549 cells were grown and treated with oligonucleotide (300 nM) as described in Example 2. Cells were harvested at 24, 48 and 72 hours after treatment. RhoA protein levels were measured by Western blotting. After oligonucleotide treatment, cells were washed twice in phosphate-buffered saline (PBS) and lysed in 25 mM Tris-HCl pH 7.5, 1% Triton X-100, 0.2% SDS, 0.5% sodium deoxycholate, 450 mM NaCl, and 10 mg/ml aprotinin and leupeptin. After 15 minutes on ice, the samples were centrifuged at maximum speed in a microfuge. Protein concentration was determined by Bradford reagent (Bio-Rad Laboratories, Hercules, Calif.). Fifty mg of protein was separated by SDS-PAGE (15%). Following electrophoresis, proteins were transferred to IMMOBILON-P membranes (Millipore, Bedford, Mass.) The membrane was blocked in 5% fish gelatin (Sigma Chemicals, St. Louis, Mo.) and RhoA specific antibodies were used to visualize the proteins. After incubation with the appropriate secondary antibody, proteins were visualized using either LUMIGLO Reagent (New England Biolabs, Beverly, Mass.) or ECL PLUS (Amersham Pharmacia Biotech, Piscataway, N.J.) . Inhibition of RhoA protein was observable after 24 hours. After 48 hours, RhoA protein concentration was reduced by 80% using 17131 (SEQ ID NO. 14). Minimal inhibition was seen with 17163 (SEQ ID NO. 190), an oligonucleotide targeted to Rac1. Results are shown in Table 7. TABLE 7 Time course of RhoA Antisense Oligonucleotides (ASOs) in A549 Cells Time SEQ ID ASO Gene after % protein % protein ISIS # NO: Target treatment Expression Inhibition control — LIPOFECTIN —  100%   0% only 17131 14 3′-UTR 24 hr 46.2% 53.8% 17131 14 ″ 48 hr 16.0% 84.0% 17131 14 ″ 72 hr 12.4% 87.6% 17163 190 Rac1 control 24 hr 104.1%  — 17163 190 ″ 48 hr 82.3% 17.7% 17163 190 ″ 72 hr 95.2%  4.8%

Example 6 Dose Response of Antisense Oligonucleotide Effects on Human RhoA Protein Levels in A549 Cells

[0113] Oligonucleotide 17131 was tested for a dose response by using varying concentrations of oligonucleotide and measuring the effect of the oligonucleotide on RhoA protein levels. A549 cells were grown and treated with oligonucleotide (concentrations indicated in Table 8) as described in Example 2. Western blotting was performed to measure protein levels as described in Example 5. A dose response effect is seen with 17131 (SEQ ID NO. 14), whereas the scrambled control 18550 (SEQ ID NO. 19) had no effect on RhoA protein levels. TABLE 8 Dose response of RhoA antisense oligonucleotide on protein levels in A549 cells SEQ ID ASO Gene % protein % protein ISIS # NO: Target Dose Expression Inhibition control — LIPOFECTIN — 100%  0% only 17131 14 3′-UTR  75 nM  51% 49% 17131 14 ″ 150 nM  23% 77% 17131 14 ″ 300 nM  20% 80% 18550 19 control  75 nM 101% — 18550 19 ″ 150 nM 101% — 18550 19 ″ 300 nM 104% —

Example 7 Inhibition of JNK Activation by RhoA Antisense Oligonucleotides in A549 Cells Stimulated with H₂O₂

[0114] Oligonucleotide 17131 (SEQ ID NO. 14) was tested for its ability to inhibit JNK activation by stimulation with H₂O₂ or Il-1b. A549 cells were grown as described in Example 2. Cells were treated with 150 nM of oligonucleotide for four hours. After treatment, the media was replaced with DMEM, 0.1% FCS, and the cells were left in culture for 48 hours prior to stimulation. Stimulation was with either 30 ng/ml IL-1b or 1 mM H₂O₂ for 30 minutes. After stimulation, the cells were washed twice in PBS, and lysed in 25 mM Hepes pH 7.7, 0.3 M NaCl, 1.5 mM MgCl₂, 0.1% Triton X-100, 20 mM b-glycerophosphate, 0.1 mM sodium orthovanadate (Na₃VO₄), 0.5 mM PMSF, and 10 mg/ml of aprotinin and leupeptin. After 20 minutes on ice, the lysates were centrifuged at maximum speed in a microfuge for 20 minutes. The protein concentration in the supernatant was determined using Bradford reagent (Bio-Rad Laboratories, Hercules, Calif.). To 150 mg of protein, 25 ml of c-Jun fusion beads (New England Biolabs, Beverly, Mass.) were added and incubated at 4° C. on a rotating wheel overnight. The samples were then washed four times in 20 mM Hepes pH 7.7, 50 mM NaCl, 0.1 mM EDTA, 2.5 mM MgCl₂, and 0.05% Triton X-100 (HIBI buffer). The kinase reaction was run for 20 minutes at 30° C. in 20 mM Hepes pH 7.7, 20 mM MgCl₂, 20 mM b-glycerophosphate, 20 mM p-nitrophenyl phosphate, 0.1 mM Na₃VO₄, 2 mM DTT, 20 mM ATP, and 5 mCi of g[³²P]-ATP. The reaction was stopped with 500 ml of ice cold HIBI buffer. The beads were pelleted, resuspended in PAGE loading buffer, boiled for 5 minutes, and the products separated on a 12% SDS gel (Novex, La Jolla, Calif.). Bands were quantitated using a PhosphorImager.

[0115] Results are shown in Table 9. Oligonucleotide 17131 (SEQ ID NO. 14) showed moderate but specific inhibition of H₂O₂-induced JNK activation. TABLE 9 Inhibition of JNK activation by RhoA antisense oligonucleotides SEQ ID ASO Gene % inhibition % inhibition ISIS # NO: Target Dose Il-1b induced H₂O₂ induced control 13 LIPOFECTIN —   0%   0% only 17131 14 3′-UTR 150 nM — 37.6% 18550 19 control 150 nM 2.2%  5.8%

Example 8 Synthesis of Additional RhoA Sequences

[0116] Additional oligonucleotides were synthesized in 96 well plate format via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a standard 96 well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile-. Standard base-protected beta-cyanoethyl-di-isopropyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per published methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.

[0117] Oligonucleotides were cleaved from support and deprotected with concentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

[0118] A series of oligonucleotides were designed to target different regions of the human RhoA RNA, using published sequences (GenBank accession number X05026, incorporated herein as SEQ ID NO: 1). The oligonucleotides are shown in Table 10. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. X05026), to which the oligonucleotide binds.

[0119] All compounds in Table 10 are oligodeoxynucleotides with phosphorothioate backbones (internucleoside linkages) throughout. All compounds in Table 11 are chimeric oligonucleotides (“gapmers”) 18 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by four-nucleotide “wings.” The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. Cytidine residues in the 2′-MOE wings are 5-methylcytidines. TABLE 10 Nucleotide Sequences of Human RhoA Phosphorothioate Oligodeoxynucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET NO. (5′ -> 3′) NO: CO-ORDINATES¹ REGION 25544 AGAGAACCGACGGAGCAC 23 0030-0047 5′-UTR 25545 GTCGACTAATGAGAGAAC 24 0041-0058 5′-UTR 25546 GACCGTCCACTAATCAGA 25 0045-0062 5′-UTR 25547 AGCTGAAGACCAGACCGT 26 0057-0074 5′-UTR 25548 AGTCGCAAACTCGGAGAC 4 0085-0102 5′-UTR 25549 AATCCGAGTCCAGCCTCT 27 0128-0145 5′-UTR 25550 AACGAATCCGAGTCCACC 28 0132-0149 5′-UTR 25551 TCAGGCAACGAATCCCAG 29 0138-0155 5′-UTR 25552 CACCAACAATCACCAGTT 30 0178-0195 Coding 25553 AAGACTATGAGCAAGCAT 31 0215-0232 Coding 25554 ATACACCTCTGGGAACTG 32 0243-0260 Coding 25555 ACATAGTTCTCAAACACT 33 0269-0286 Coding 25556 ACTCTACCTGCTTTCCAT 34 0304-0321 Coding 25557 CACAAACCCAACTCTACC 35 0314-0331 Coding 25558 AACATCGCTATCTGGGTA 36 0378-0395 Coding 25559 TTCTGGGATGTTTTCTAA 37 0432-0449 Coding 25560 GGACAGAAATGCTTGACT 38 0464-0481 Coding 25561 GTGCTCATCATTCCGAAG 39 0519-0536 Coding 25562 CTTCTCTGCTCATCATTC 40 0524-0541 Coding 25563 TAGCTCCCGCCTTGTGTG 41 0534-0551 Coding 25564 CCAATCCTGTTTGCCATA 42 0596-0613 Coding 25565 GTCTTTCCTGAACACTCC 43 0629-0646 Coding 25566 AAAACCTCTCTCACTCCA 44 0653-0670 Coding 25567 AAGACAAGGCAACCAGAT 45 0719-0736 Coding 25568 TTTCACAAGACAAGGCAA 46 0725-0742 Stop 25569 GCAAGGTTTCACAAGACA 47 0731-0748 Stop 25570 ATTAACCGCATAAGGGCT 48 0758-0775 3′-UTR 25571 TAATAAACAGCACTTCAA 49 0777-0794 3′-UTR 25572 CCAGTAATCATACACTAA 50 0798-0815 3′-UTR 25573 ATGACTTCTGATTTGTAA 51 0847-0864 3′-UTR 25574 TAGCAAGATGACTTCTGA 52 0854-0871 3′-UTR 25575 CTGGTAGOAAGATGACTT 53 0858-0875 3′-UTR 25576 CTAAATACTGGTAGCAAG 54 0865-0882 3′-UTR 25577 TTGGCTTCTAAATACTGG 55 0872-0889 3′-UTR 25578 TCATAGTTGGCTTCTAAA 56 0878-0895 3′-UTR 25579 AATAATCATAGTTGGCTT 57 0883-0900 3′-UTR 25580 TCAAAAGGACCCTGGTGG 58 0923-0940 3′-UTR 25581 GTGCAGAGGAGGGCTGTT 59 0950-0967 3′-UTR 25582 CCAACTGTTTCTCTTTCT 60 1026-1043 3′-UTR 25583 AAGTAGTTACAGCCTAAT 61 1056-1073 3′-UTR

Example 9 Total RNA Isolation

[0120] Total mRNA was isolated using an RNEASY 96 kit and buffers purchased from Qiagen Inc. (Valencia Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 100 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 100 μL of 70% ethanol was then added to each well and the contents mixed by pippeting three times up and down. The samples were then transferred to the RNEASY 96 well plate attached to a QIAVAC manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL of Buffer RW1 was added to each well of the RNEASY 96 plate and the vacuum again applied for 15 seconds. 1 mL of Buffer RPE was then added to each well of the RNEASY 96 plate and the vacuum applied for a period of 15 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 10 minutes. The plate was then removed from the QIAVAC manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 60 μL water into each well, incubating 1 minute, and then applying the vacuum for 30 seconds. The elution step was repeated with an additional 60 μL water.

[0121] Poly(A)+ mRNA may be isolated according to Miura et al., Clin. Chem., 42, 1758 (1996). Other methods for poly(A)+ mRNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., (1993). Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 ml cold PBS. 60 ml lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 ml of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 ml of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 ml of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C. was added to each well, the plate was incubated on a 90° hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.

[0122] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

Example 10 Real-Time Quantitative PCR Analysis of RhoA mRNA Levels

[0123] Quantitation of RhoA mRNA levels was determined by real-time quantitative PCR using the ABI PRISM 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR, in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., JOE or FAM, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular (six-second) intervals by laser optics built into the ABI PRISM 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.

[0124] PCR reagents were obtained from PE-Applied Biosystems, Foster City, Calif.. RT-PCR reactions were carried out by adding 25 μL PCR cocktail (1×TAQMAN buffer A, 5.5 mM MgCl₂, 300 μM each of dATP, dCTP and dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer, and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD, and 12.5 Units MuLV reverse transcriptase) to 96 well plates containing 25 μL poly(A) mRNA solution. The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the AMPLITAQ GOLD, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension). RhoA probes and primers were designed to hybridize to the human RhoA sequence, using published sequence information (GenBank accession number X05026, incorporated herein as SEQ ID NO: 1). For RhoA the PCR primers were: forward primer: GGCTGGACTCGGATTCGTT (SEQ ID NO: 62) reverse primer: CCATCACCAACAATCACCAGTT (SEQ ID NO: 63) and the PCR probe was: FAM-CCTGAGCAATGGCTGCCATCCG-TAMRA

[0125] (SEQ ID NO: 64) where FAM (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye. For GAPDH the PCR primers were: forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 65) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 66) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC- TAMPA 3′ (SEQ ID NO: 67)

[0126] ID NO: 67) where JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

Example 11 Antisense Inhibition of RhoA Expression-Phosphorothioate Oligodeoxynucleotides

[0127] In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human RhoA RNA, using published sequences (GenBank accession number X05026, incorporated herein as SEQ ID NO: 1). The oligonucleotides are shown in Table 10. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. X05026), to which the oligonucleotide binds. All compounds in Table 10 are oligodeoxynucleotides with phosphorothioate backbones (internucleoside linkages) throughout. The compounds were analyzed for effect on RhoA mRNA levels by quantitative real-time PCR as described in other examples herein. Data are shown in Table 11 and are averages from three experiments. If present, “N.D.” indicates “no data”. TABLE 11 Inhibition of RhoA mRNA levels by phosphorothioate oligodeoxynucleotides SEQ TARGET % Inhi- ID ISIS# REGION SITE SEQUENCE bition NO. 25544 5′ UTR 30 AGACAACCGACGGAGGAC 47 23 25545 5′ UTR 41 GTGGACTAATGAGAGAAC 0 24 25546 5′ UTR 45 GACCGTGGACTAATGAGA 40 25 25547 5′ UTR 57 AGCTGAAGACCAGACCGT 76 26 25548 5′ UTR 85 AGTCGCAAACTCGGAGAC 36 4 25549 5′ UTR 128 AATCCGAGTCCAGCCTCT 67 27 25550 5′ UTR 132 AACGAATCCGAGTCCAGC 34 28 25551 5′ UTR 138 TCAGGCAACGAATCCGAG 59 29 25552 CODING 178 CACCAACAATCACCAGTT 47 30 25553 CODING 215 AAGACTATGAGCAAGCAT 36 31 25554 CODING 243 ATACACCTCTGGGAACTG 74 32 25555 CODING 269 ACATAGTTCTCAAACACT 31 33 25556 CODING 304 ACTCTACCTGCTTTCCAT 64 34 25557 CODING 314 CACAAAGCCAACTCTACC 25 35 25558 CODING 378 AACATCGGTATCTGGGTA 35 36 25559 CODING 432 TTCTGGGATGTTTTCTAA 21 37 25560 CODING 464 GGACAGAAATGCTTGACT 64 38 25561 CODING 519 GTGCTCATCATTCCGAAG 71 39 25562 CODING 524 CTTGTGTGCTCATCATTC 38 40 25563 CODING 534 TAGCTCCCGCCTTGTGTG 78 41 25564 CODING 596 CCAATCCTGTTTGCCATA 82 42 25565 CODING 629 GTCTTTGCTGAACACTCC 56 43 25566 CODING 653 AAAACCTCTCTCACTCCA 68 44 25567 CODING 719 AAGACAAGGCAACCAGAT 55 45 25568 STOP 725 TTTCACAAGACAAGGCAA 0 46 25569 STOP 731 GCAAGGTTTCACAAGACA 37 47 25570 3′ UTR 758 ATTAACCGCATAAGGGCT 77 48 25571 3′ UTR 777 TAATAAACAGCACTTCAA 19 49 25572 3′ UTR 798 CCAGTAATCATACACTAA 26 50 25573 3′ UTR 847 ATGACTTCTGATTTGTAA 27 51 25574 3′ UTR 854 TAGCAAGATGACTTCTGA 62 52 25575 3′ UTR 858 CTGGTAGCAAGATGACTT 59 53 25576 3′ UTR 865 CTAAATACTGGTAGCAAG 29 54 25577 3′ UTR 872 TTGGCTTCTAAATACTGG 57 55 25578 3′ UTR 878 TCATAGTTGGCTTCTAAA 60 56 25579 3′ UTR 883 AATAATCATAGTTGGCTT 33 57 25580 3′ UTR 923 TCAAAAGGACCCTGGTGG 25 58 25581 3′ UTR 950 GTGCAGAGGAGGGCTGTT 68 59 25582 3′ UTR 1026 CCAACTGTTTCTCTTTCT 52 60 25583 3′ UTR 1056 AAGTAGTTACAGCCTAAT 26 61

[0128] As shown in Table 11, SEQ ID NOs 23, 26, 27, 29, 30, 32, 34, 38, 39, 41, 42, 43, 44, 45, 48, 52, 53, 56, 57, 59 and 60 demonstrated at least 45% inhibition of RhoA expression in this assay and are therefore preferred.

Example 12 Antisense Inhibition of RhoA Expression-Phosphorothioate 2′-MOE Gapmer Oligonucleotides

[0129] In accordance with the present invention, a second series of oligonucleotides targeted to human RhoA were synthesized. The oligonucleotide sequences are shown in Table 12. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. X05026), to which the oligonucleotide binds.

[0130] All compounds in Table 12 are chimeric oligonucleotides (“gapmers”) 18 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by four-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. Cytidine residues in the 2′-MOE wings are 5-methylcytidines. TABLE 12 Nucleotide Sequences of Human RhoA Gapmer Oligonucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET NO. (5′ -> 3′) NO: CO-ORDINATES¹ REGION 25584 AGAGAACCGACGGAGGAC 23 0030-0047 5′-UTR 25585 GTGGACTAATGAGAGAAC 24 0041-0058 5′-UTR 25586 GACCGTGGACTAATGAGA 25 0045-0062 5′-UTR 25587 AGCTGAAGACCAGACCGT 26 0057-0074 5′-UTR 25588 AGTCGCAAACTCGGAGAC 4 0085-0102 5′-UTR 25589 AATCCGAGTCCAGCCTCT 27 0128-0145 5′-UTR 25590 AACGAATCCGAGTCCAGC 28 0132-0149 5′-UTR 25591 TCAGGCAACGAATCCGAG 29 0138-0155 5′-UTR 25592 CACCAACAATCACCAGTT 30 0178-0195 Coding 25593 AAGACTATGAGCAAGCAT 31 0215-0232 Coding 25594 ATACACCTCTGGGAACTG 32 0243-0260 Coding 25595 ACATAGTTCTCAAACACT 33 0269-0286 Coding 25596 ACTCTACCTGCTTTCCAT 34 0304-0321 Coding 25597 CACAAAGCCAACTCTACC 35 0314-0331 Coding 25598 AACATCGGTATCTGGGTA 36 0378-0395 Coding 25599 TTCTGGGATGTTTTCTAA 37 0432-0449 Coding 25600 GGACAGAAATGCTTGACT 38 0464-0481 Coding 25601 GTGCTCATCATTCCGAAG 39 0519-0536 Coding 25602 CTTGTGTGCTCATCATTC 40 0524-0541 Coding 25603 TAGCTCCCGCCTTGTGTG 41 0534-0551 Coding 25604 CCAATCCTGTTTGCCATA 42 0596-0613 Coding 25605 GTCTTTGCTGAACACTCC 43 0629-0646 Coding 25606 AAAACCTCTCTCACTCCA 44 0653-0670 Coding 25607 AAGACAACCCAACCAGAT 45 0719-0736 Coding 25608 TTTCACAAGACAAGGCAA 46 0725-0742 Stop 25609 GCAACCTTTCACAAGACA 47 0731-0748 Stop 25610 ATTAACCCCATAACGGCT 48 0758-0775 3′-UTR 25611 TAATAAACAGCACTTCAA 49 0777-0794 3′-UTR 25612 CCAGTAATCATACACTAA 50 0798-0815 3′-UTR 25613 ATGACTTCTGATTTGTAA 51 0847-0864 3′-UTR 25614 TAGCAAGATGACTTCTGA 52 0854-0871 3′-UTR 25615 CTGGTAGCAAGATGACTT 53 0858-0875 3′-UTR 25616 CTAAATACTGGTAGCAAG 54 0865-0882 3′-UTR 25617 TTGGCTTCTAAATACTGG 55 0872-0889 3′-UTR 25618 TCATAGTTCGCTTCTAAA 56 0878-0895 3′-UTR 25619 AATAATCATAGTTGGCTT 57 0883-0900 3′-UTR 25620 TCAAAAGGACCCTCGTGG 58 0923-0940 3′-UTR 25621 GTGCAGAGGAGGCCTGTT 59 0950-0967 3′-UTR 25622 CCAACTCTTTCTCTTTCT 60 1026-1043 3′-UTR 25623 AAGTAGTTACAGCCTAAT 61 1056-1073 3′-UTR

[0131] The oligonucleotides shown in Table 12 were tested by real-time quantitative PCR as described in other examples herein and data are shown in Table 13 (averaged from three experiments). If present, “N.D.” indicates “no data”. TABLE 13 Inhibition of RhoA mRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap SEQ TARGET % Inhi- ID ISIS# REGION SITE SEQUENCE bition NO. 25584 5′ UTR 30 AGAGAACCGACGGAGGAC 44 23 25585 5′ UTR 41 GTGGACTAATGAGAGAAC 35 24 25586 5′ UTR 45 GACCGTGGACTAATGAGA 53 25 25587 5′ UTR 57 AGCTGAAGACCAGACCGT 62 26 25588 5′ UTR 85 AGTCGCAAACTCGGAGAC 54 4 25589 5′ UTR 128 AATCCGAGTCCAGCCTCT 38 27 25590 5′ UTR 132 AACGAATCCGAGTCCAGC 47 28 25591 5′ UTR 138 TCAGGCAACGAATCCGAG 31 29 25592 CODING 178 CACCAACAATCACCAGTT 0 30 25593 CODING 215 AAGACTATGAGCAAGCAT 43 31 25594 CODING 243 ATACACCTCTGGGAACTG 23 32 25595 CODING 269 ACATAGTTCTCAAACACT 16 33 25596 CODING 304 ACTCTACCTGCTTTCCAT 0 34 25597 CODING 314 CACAAAGCCAACTCTACC 0 35 25598 CODING 378 AACATCGGTATCTGGGTA 65 36 25599 CODING 432 TTCTGGGATGTTTTCTAA 53 37 25600 CODING 464 GGACAGAAATGCTTGACT 50 38 25601 CODING 519 GTGCTCATCATTCCGAAG 45 39 25602 CODING 524 CTTGTGTGCTCATCATTC 26 40 25603 CODING 534 TAGCTCCCGCCTTGTGTG 59 41 25604 CODING 596 CCAATCCTGTTTGCCATA 40 42 25605 CODING 629 GTCTTTGCTGAACACTCC 47 43 25606 CODING 653 AAAACCTCTCTCACTCCA 30 44 25607 CODING 719 AAGACAAGGCAACCAGAT 0 45 25608 STOP 725 TTTCACAAGACAAGGCAA 7 46 25609 STOP 731 GCAAGGTTTCACAAGACA 53 47 25610 3′ UTR 758 ATTAACCGCATAAGGGCT 56 48 25611 3′ UTR 777 TAATAAACAGCACTTCAA 7 49 25612 3′ UTR 798 CCAGTAATCATACACTAA 41 50 25613 3′ UTR 847 ATGACTTCTGATTTGTAA 53 51 25614 3′ UTR 854 TAGCAAGATGACTTCTGA 59 52 25615 3′ UTR 858 CTGGTAGCAAGATGACTT 67 53 25616 3′ UTR 865 CTAAATACTGGTAGCAAG 65 54 25617 3′ UTR 872 TTGGCTTCTAAATACTGG 74 55 25618 3′ UTR 878 TCATAGTTGGCTTCTAAA 52 56 25619 3′ UTR 883 AATAATCATACTTGGCTT 49 57 25620 3′ UTR 923 TCAAAAGGACCCTGGTGG 58 58 25621 3′ UTR 950 GTGCAGAGGAGGGCTGTT 60 59 25622 3′ UTR 1026 CCAACTGTTTCTCTTTCT 62 60 25623 3′ UTR 1056 AAGTAGTTACACCCTAAT 44 61

[0132] As shown in Table 13, SEQ ID NOs 23, 24, 25, 26, 4, 27, 28, 31, 36, 37, 38, 39, 41, 42, 43, 47, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 and 61 demonstrated at least 35% inhibition of RhoA expression in this experiment and are therefore preferred.

Example 13 Synthesis of RhoB Antisense Oligonucleotide Sequences

[0133] Oligonucleotide sequences were synthesized as described in previous examples. Antisense oligonucleotides were designed to target human RhoB. Target sequence data are from the RhoB cDNA sequence published by Chardin, P., et al. (Nucleic Acids Res. 1988, 16, 2717); Genbank accession number X06820, provided herein as SEQ ID NO: 68. TABLE 14 Nucleotide Sequences of Human RhoB Phosphorothioate Oligodeoxynucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET NO. (5′ -> 3′) NO: CO-ORDINATES¹ REGION 25384 CCACCACCACCTTCTTCC 69 0014-0031 Coding 25385 CCCTCCCCCACCACCACC 70 0024-0041 Coding 25386 GCACGTCTTGCCACACGC 71 0043-0060 Coding 25387 ACTGAACACGATCAGCAG 72 0061-0078 Coding 25388 TTACTGAACACGATCAGC 73 0063-0080 Coding 25389 CCTTACTGAACACGATCA 74 0065-0082 Coding 25390 GTCCTTACTGAACACGAT 75 0067-0084 Coding 25391 CTCGTCCTTACTGAACAC 76 0070-0087 Coding 25392 AACTCGTCCTTACTGAAC 77 0072-0089 Coding 25393 CATAGTTCTCGAAGACGG 78 0110-0127 Coding 25394 TCGCCCACATAGTTCTCG 79 0117-0134 Coding 25395 CCGTCCACCTCAATGTCG 80 0132-0149 Coding 25396 AAGCACATGAGAATGACG 81 0234-0251 Coding 25397 GAGTCCGGGCTGTCCACC 82 0255-0272 Coding 25398 ATGTTCTCCAGCGAGTCC 83 0267-0284 Coding 25399 GGGATGTTCTCCAGCGAG 84 0270-0287 Coding 25400 GACATGCTCGTCGCTGCG 85 0364-0381 Coding 25401 CCGACATGCTCGTCGCTG 86 0366-0383 Coding 25402 TGTGCGGACATGCTCGTC 87 0370-0387 Coding 25403 CTCTGTGCGGACATGCTC 88 0373-0390 Coding 25404 CCAGCTCTGTGCGGACAT 89 0377-0394 Coding 25405 CGCCCCACCTCTGTGCGG 90 0381-0398 Coding 25406 TGCGGGCCAGCTCTGTGC 91 0383-0400 Coding 25407 GTTCCTGCTTCATGCGGG 92 0395-0412 Coding 25408 ACGGGTTCCTGCTTCATG 93 0399-0416 Coding 25409 GTAGTCGTAGGCTTGGAT 94 0451-0468 Coding 25410 CGACGTAGTCGTAGCCTT 95 0455-0472 Coding 25411 GTCTTCCCACACCACTCG 96 0471-0488 Coding 25412 ACCTCGCGCACGCCTTCC 97 0492-0509 Coding 25413 AGACCTCGCGCACGCCTT 98 0494-0511 Coding 25414 CGAAGACCTCCCGCACGC 99 0497-0514 Coding 25415 CTCGAAGACCTCGCGCAC 100 0499-0516 Coding 25416 GCCGTCTCGAAGACCTCG 101 0504-0521 Coding 25417 CCTGGCCGTCTCGAACAC 102 0508-0525 Coding 25418 GTTCTGGGAGCCGTAGCG 103 0544-0561 Coding 25419 GCCGTTCTGGGAGCCGTA 104 0547-0564 Coding 25420 GATGCAGCCGTTCTGGGA 105 0553-0570 Coding 25421 GTTGATGCAGCCGTTCTG 106 0556-0573 Coding 25422 CAGCAGTTGATCCAGCCG 107 0561-0578 Coding 25423 AGCACCTTGCAGCAGTTG 108 0570-0587 Coding

Example 14 Antisense Inhibition of RhoB Expression-Phosphorothioate Oligodeoxynucleotides

[0134] In accordance with the present invention, the oligonucleotides shown in Table 14 were analyzed for effect on RhoB mRNA levels by quantitative real-time PCR as described in examples herein. Data are averages from three experiments. If present, “N.D.” indicates “no data”. TABLE 15 Inhibition of RhoB mRNA levels by phosphorothioate oligodeoxynucleotides SEQ TARGET % Inhibi- ID ISIS# REGION SITE SEQUENCE tion NO. 25384 Coding 14 CCACCACCAGCTTCTTGC 0  69 25385 CODING 24 CCGTCGCCCACCACCACC 0  70 25386 CODING 43 GCACGTCTTGCCACACGC 0  71 25387 CODING 61 ACTGAACACGATCAGCAG 0  72 25388 CODING 63 TTACTGAACACGATCAGC 0  73 25389 CODING 65 CCTTACTGAACACGATCA 0  74 25390 CODING 67 GTCCTTACTGAACACGAT 5  75 25391 CODING 70 CTCGTCCTTACTGAACAC 1  76 25392 CODING 72 AACTCGTCCTTACTGAAC 30  77 25393 CODING 110 CATAGTTCTCGAAGACGG 0  78 25394 CODING 117 TCGGCCACATAGTTCTCG 13  79 25395 CODING 132 CCGTCCACCTCAATGTCG 0  80 25396 CODING 234 AAGCACATGAGAATGACG 0  81 25397 CODING 255 GAGTCCGGGCTGTCCACC 0  82 25398 CODING 267 ATGTTCTCCAGCGAGTCC 0  83 25399 CODING 270 GGGATGTTCTCCAGCGAG 33  84 25400 CODING 364 GACATGCTCGTCGCTGCG 0  85 25401 CODING 366 CGGACATGCTCGTCGCTG 0  86 25402 CODING 370 TGTGCGGACATGCTCGTC 0  87 25403 CODING 373 CTCTGTGCGGACATGCTC 39  88 25404 CODING 377 CCAGCTCTGTGCGGACAT 21  89 25405 CODING 381 CGGGCCAGCTCTGTGCGG 38  90 25406 CODING 383 TGCGGGCCAGCTCTGTGC 31  91 25407 CODING 395 GTTCCTGCTTCATGCGGG 27  92 25408 CODING 399 ACGGGTTCCTGCTTCATG 0  93 25409 CODING 451 GTAGTCGTAGGCTTGGAT 29  94 25410 CODING 455 CGAGGTAGTCGTAGGCTT 39  95 25411 CODING 471 GTCTTGGCAGAGCACTCG 20  96 25412 CODING 492 ACCTCGCGCACGCCTTCC 0  97 25413 CODING 494 AGACCTCGCGCACGCCTT 16  98 25414 CODING 497 CGAAGACCTCGCGCACGC 0  99 25415 CODING 499 CTCGAAGACCTCGCGCAC 0 100 25416 CODING 504 GCCGTCTCGAAGACCTCG 0 101 25417 CODING 508 CGTGGCCGTCTCGAAGAC 0 102 25418 CODING 544 GTTCTGGGAGCCGTAGCG 36 103 25419 CODING 547 GCCGTTCTGGGAGCCGTA 0 104 25420 CODING 553 GATGCAGCCGTTCTGGGA 0 105 25421 CODING 556 GTTGATGCAGCCGTTCTG 7 106 25422 CODING 561 CAGCAGTTGATGCAGCCG 31 107 25423 CODING 570 AGCACCTTGCAGCAGTTG 0 108

[0135] As shown in Table 15, SEQ ID Nos 77, 84, 88, 90, 91, 92, 94, 95, 103 and 107 demonstrated at least 25% inhibition of RhoB expression in this assay and are therefore preferred.

Example 15 Antisense Inhibition of RhoB Expression-Phosphorothioate 2′-MOE Gapmer Oligonucleotides

[0136] In accordance with the present invention, a second series of oligonucleotides targeted to human RhoB were synthesized. The oligonucleotide sequences are shown in Table 16. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. X06820), to which the oligonucleotide binds.

[0137] All compounds in Table 16 are chimeric oligonucleotides (“gapmers”) 18 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by four-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. Cytidine residues in the 2′-MOE wings are 5-methylcytidines. TABLE 16 Nucleotide Sequences of Human RhoB Gapmer Oligonucleotides NUCLEOTIDE TARGET GENE GENE ISIS SEQUENCE SEQ ID NUCLEOTIDE TARGET NO. (5′→3′) NO: CO-ORDINATES¹ REGION 25424 CCACCACCAGCTTCTTGC 69 0014-0031 Coding 25425 CCGTCGCCCACCACCACC 70 0024-0041 Coding 25426 GCACGTCTTGCCACACGC 71 0043-0060 Coding 25427 ACTGAACACGATCAGCAG 72 0061-0078 Coding 25428 TTACTGAACACGATCAGC 73 0063-0080 Coding 25429 CCTTACTGAACACGATCA 74 0065-0082 Coding 25430 GTCCTTACTGAACACGAT 75 0067-0084 Coding 25431 CTCGTCCTTACTGAACAC 76 0070-0087 Coding 25432 AACTCGTCCTTACTGAAC 77 0072-0089 Coding 25433 CATAGTTCTCGAAGACGG 78 0110-0127 Coding 25434 TCGGCCACATAGTTCTCG 79 0117-0134 Coding 25435 CCGTCCACCTCAATGTCG 80 0132-0149 Coding 25436 AAGCACATGAGAATGACG 81 0234-0251 Coding 25437 GAGTCCGGGCTGTCCACC 82 0255-0272 Coding 25438 ATGTTCTCCAGCGAGTCC 83 0267-0284 Coding 25439 GGGATGTTCTCCAGCGAG 84 0270-0287 Coding 25440 GACATGCTCGTCGCTGCG 85 0364-0381 Coding 25441 CGGACATGCTCGTCGCTG 86 0366-0383 Coding 25442 TGTGCGGACATGCTCGTC 87 0370-0387 Coding 25443 CTCTGTGCGGACATGCTC 88 0373-0390 Coding 25444 CCAGCTCTGTGCGGACAT 89 0377-0394 Coding 25445 CGGGCCAGCTCTGTGCGG 90 0381-0393 Coding 25446 TGCGGGCCAGCTCTGTGC 91 0383-0400 Coding 25447 GTTCCTGCTTCATGCGGG 92 0395-0412 Coding 25448 ACGGGTTCCTCCTTCATG 93 0399-0416 Coding 25449 GTAGTCGTAGGCTTGGAT 94 0451-0468 Coding 25450 CGAGGTAGTCGTAGGCTT 95 0455-0472 Coding 25451 GTCTTGGCAGAGCACTCG 96 0471-0488 Coding 25452 ACCTCGCGCACGCCTTCC 97 0492-0509 Coding 25453 AGACCTCGCGCACGCCTT 98 0494-0511 Coding 25454 CGAAGACCTCGCGCACGC 99 0497-0514 Coding 25455 CTCGAAGACCTCGCGCAC 100 0499-0516 Coding 25456 GCCGTCTCGAAGACCTCG 101 0504-0521 Coding 25457 CGTGGCCGTCTCGAAGAC 102 0508-0525 Coding 25458 GTTCTGGGAGCCGTAGCG 103 0544-0561 Coding 25459 GCCGTTCTGGGAGCCGTA 104 0547-0564 Coding 25460 GATGCACCCGTTCTGGGA 105 0553-0570 Coding 25461 GTTGATGCAGCCGTTCTG 106 0556-0573 Coding 25462 CAGCAGTTGATGCAGCCG 107 0561-0578 Coding 25463 AGCACCTTCCACCAGTTG 108 0570-0587 Coding

[0138] Data for the compounds in Table 16 were obtained by real-time quantitative PCR as described in other examples herein and are averaged from three experiments. Results are shown in Table 17. If present, “N.D.” indicates “no data”. TABLE 17 Inhibition of RhoB mRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap % TARGET Inhi- SEQ ID ISIS# REGION SITE SEQUENCE bition NO. 25424 Coding 14 CCACCACCAGCTTCTTGC 29 69 25425 CODING 24 CCGTCGCCCACCACCACC 23 70 25426 CODING 43 GCACGTCTTGCCACACGC 46 71 25427 CODING 61 ACTGAACACGATCAGCAG 37 72 25428 CODING 63 TTACTGAACACGATCAGC 47 73 25429 CODING 65 CCTTACTGAACACGATCA 7 74 25430 CODING 67 GTCCTTACTGAACACGAT 46 75 25431 CODING 70 CTCGTCCTTACTGAACAC 52 76 25432 CODING 72 AACTCGTCCTTACTGAAC 35 77 25433 CODING 110 CATAGTTCTCGAAGACGG 29 78 25434 CODING 117 TCGGCCACATAGTTCTCG 65 79 25435 CODING 132 CCGTCCACCTCAATGTCG 40 80 25436 CODING 234 AAGCACATGAGAATGACG 44 81 25437 CODING 255 GAGTCCGGGCTGTCCACC 36 82 25438 CODING 267 ATGTTCTCCAGCGAGTCC 28 83 25439 CODING 270 GGGATGTTCTCCAGCGAG 54 84 25440 CODING 364 GACATGCTCGTCGCTGCG 49 85 25441 CODING 366 CGGACATGCTCGTCGCTG 46 86 25442 CODING 370 TGTGCGGACATGCTCGTC 65 87 25443 CODING 373 CTCTGTGCGGACATGCTC 39 88 25444 CODING 377 CCAGCTCTGTGCGGACAT 19 89 25445 CODING 381 CGGGCCAGCTCTGTGCGG 21 90 25446 CODING 383 TGCGGGCCAGCTCTGTGC 9 91 25447 CODING 395 GTTCCTGCTTCATGCGGG 16 92 25448 CODING 399 ACGGGTTCCTGCTTCATG 7 93 25449 CODING 451 GTAGTCGTAGGCTTGGAT 38 94 25450 CODING 455 CGAGGTAGTCGTAGGCTT 0 95 25451 CODING 471 GTCTTGGCAGAGCACTCG 42 96 25452 CODING 492 ACCTCGCGCACGCCTTCC 9 97 25453 CODING 494 AGACCTCGCGCACGCCTT 7 98 25454 CODING 497 CGAAGACCTCGCGCACGC 12 99 25455 CODING 499 CTCGAAGACCTCGCGCAC 23 100 25456 CODING 504 GCCGTCTCGAAGACCTCG 34 101 25457 CODING 508 CGTGGCCGTCTCGAAGAC 27 102 25458 CODING 544 GTTCTGGGAGCCGTAGCG 58 103 25459 CODING 547 GCCGTTCTGGGAGCCGTA 63 104 25460 CODING 553 GATGCAGCCGTTCTGGGA 17 105 25461 CODING 556 GTTGATGCAGCCGTTCTG 21 106 25462 CODING 561 CAGCAGTTGATGCAGCCG 50 107 25463 CODING 570 AGCACCTTGCAGCAGTTG 55 108

[0139] As shown in Table 17, SEQ ID Nos 71, 62, 63, 75, 76, 77, 79, 80, 81, 82, 84, 85, 86, 87, 88, 94, 96, 101, 103, 104, 107 and 108 demonstrated at least 30% inhibition of RhoB expression in this experiment and are therefore preferred.

Example 16 Synthesis of RhoC Antisense Oligonucleotide Sequences

[0140] Oligonucleotide sequences were synthesized as described in previous examples. Antisense oligonucleotides were designed to target human RhoC. Target sequence data are from the RhoC cDNA sequence determined by Fagan, K. P., et al.; Genbank accession number L25081, provided herein as SEQ ID NO: 109. TABLE 18 Nucleotide Sequences of Human RhoC Phosphorothioate Oligonucleotides NUCLEOTIDE TARGET GENE GENE ISIS SEQUENCE SEQ ID NUCLEOTIDE TARGET NO. (5′→3′) NO: CO-ORDINATES¹ REGION 25304 GAGCTGAGATGAAGTCAA 110 0004-0021 5′-UTR 25305 GCTGAAGTTCCCAGGCTG 111 0044-0061 5′-UTR 25306 CCGGCTGAAGTTCCCAGG 112 0047-0064 5′-UTR 25307 GGCACCATCCCCAACGAT 113 0104-0121 Coding 25308 AGGCACCATCCCCAACGA 114 0105-0122 Coding 25309 TCCCACAGGCACCATCCC 115 0111-0128 Coding 25310 AGGTCTTCCCACAGGCAC 116 0117-0134 Coding 25311 ATGAGGAGGCACCTCTTC 117 0127-0144 Coding 25312 TTGCTGAAGACGATGAGG 118 0139-0156 Coding 25313 TCAAAGACAGTAGGGACG 119 0173-0195 Coding 25314 TTCTCAAAGACAGTAGGG 120 0181-0193 Coding 25315 ACTTCTCAAAGACAGTAG 121 0183-0200 Coding 25316 TGTTTTCCAGGCTGTCAG 122 0342-0359 Coding 25317 TCGTCTTGCCTCAGGTCC 123 0433-0450 Coding 25318 GTGTGCTCGTCTTGCCTC 124 0439-0456 Coding 25319 CTCCTGGTGTGCTCGTCT 125 0445-0462 Coding 25320 CAGACCCAACGGGCTCCT 126 0483-0500 Coding 25321 TTCCTCAGACCGAACGGG 127 0488-0505 Coding 25322 ACTCAAGGTAGCCAAAGG 128 0534-0551 Coding 25323 CTCCCGCACTCCCTCCTT 129 0566-0583 Coding 25324 CTCAAACACCTCCCGCAC 130 0575-0592 Coding 25325 GGCCATCTCAAACACCTC 131 0581-0598 Coding 25326 CTTGTTCTTGCGGACCTG 132 0614-0631 Coding 25327 CCCCTCCGACGCTTGTTC 133 0625-0642 Coding 25328 GTATGGAGCCCTCAGGAG 134 0737-0754 3′-UTR 25329 GAGCCTTCAGTATGGAGC 135 0746-0763 3′-UTR 25330 GAAAATGGAGCCTTCAGT 136 0753-0770 3′-UTR 25331 GGAACTGAAAATGGAGCC 137 0759-0776 3′-UTR 25332 GGAGGGAACTGAAAATGG 138 0763-0780 3′-UTR 25333 GCAGGAGGGAACTGAAAA 139 0766-0783 3′-UTR 25334 AGGGCAGGGCATAGGCGT 140 0851-0868 3′-UTR 25335 GGAAGGGCAGGGCATAGG 141 0854-0871 3′-UTR 25336 CATGAGGAAGGGCAGGGC 142 0859-0876 3′-UTR 25337 TAAAGTGCTGGTGTGTGA 143 0920-0937 3′-UTR 25338 CCTGTGAGCCAGAAGTGT 144 0939-0956 3′-UTR 25339 TTCCTGTGAGCCAGAAGT 145 0941-0958 3′-UTR 25340 CACTTTCCTGTGAGCCAG 146 0945-0962 3′-UTR 25341 AGACACTTTCCTGTGAGC 147 0948-0965 3′-UTR 25342 ACTCTGGGTCCCTACTGC 148 0966-0983 3′-UTR 25343 TGCAGAAACAACTCCAGG 149 0992-1009 3′-UTR

[0141] The compounds shown in Table 18 were analyzed for effect on RhoC mRNA levels by quantitative real-time PCR as described in examples herein. Data are shown in Table 19 and are averages from three experiments. If present, “N.D.” indicates “no data”. TABLE 19 Inhibition of RhoC mRNA levels by phosphorothioate oligodeoxynucleotides % TARGET Inhi- SEQ ID ISIS# REGION SITE SEQUENCE bition NO. 25304 5′ UTR 4 GAGCTGAGATGAAGTCAA 29 110 25305 5′ UTR 44 GCTGAAGTTCCCAGGCTG 25 111 25306 5′ UTR 47 CCGGCTGAAGTTCCCAGG 42 112 25307 CODING 104 GGCACCATCCCCAACGAT 81 113 25308 CODING 105 AGGCACCATCCCCAACGA 81 114 25309 CODING 111 TCCCACAGGCACCATCCC 70 115 25310 CODING 117 AGGTCTTCCCACAGGCAC 40 116 25311 CODING 127 ATGAGGAGGCAGGTCTTC 41 117 25312 CODING 139 TTGCTGAAGACGATGAGG 23 118 25313 CODING 178 TCAAAGACAGTAGGGACG 0 119 25314 CODING 181 TTCTCAAAGACAGTAGGG 2 120 25315 CODING 183 AGTTCTCAAAGACAGTAG 38 121 25316 CODING 342 TGTTTTCCAGGCTGTCAG 59 122 25317 CODING 433 TCGTCTTGCCTCAGGTCC 79 123 25318 CODING 439 GTGTGCTCGTCTTGCCTC 67 124 25319 CODING 445 CTCCTGGTGTGCTCGTCT 67 125 25320 CODING 483 CAGACCGAACGGGCTCCT 65 126 25321 CODING 488 TTCCTCAGACCGAACGGG 57 127 25322 CODING 534 ACTCAAGGTAGCCAAAGG 33 128 25323 CODING 566 CTCCCGCACTCCCTCCTT 91 129 25324 CODING 575 CTCAAACACCTCCCGCAC 34 130 25325 CODING 581 GGCCATCTCAAACACCTC 64 131 25326 CODING 614 CTTGTTCTTGCGGACCTG 72 132 25327 CODING 625 CCCCTCCGACGCTTGTTC 66 133 25328 3′ UTR 737 GTATGGAGCCCTCAGGAG 60 134 25329 3′ UTR 746 GAGCCTTCAGTATGGAGC 63 135 25330 3′ UTR 753 GAAAATGGAGCCTTCAGT 24 136 25331 3′ UTR 759 GGAACTGAAAATGGAGCC 2 137 25332 3′ UTR 763 GGAGGGAACTGAAAATGG 13 138 25333 3′ UTR 766 GCAGGAGGGAACTGAAAA 27 139 25334 3′ UTR 851 AGGGCAGGGCATAGGCGT 31 140 25335 3′ UTR 854 GGAAGGGCAGGGCATAGG 21 141 25336 3′ UTR 859 CATGAGGAAGGGCAGGGC 0 142 25337 3′ UTR 920 TAAAGTGCTGGTGTGTGA 39 143 25338 3′ UTR 939 CCTGTGAGCCAGAAGTGT 69 144 25339 3′ UTR 941 TTCCTGTGAGCCAGAAGT 69 145 25340 3′ UTR 945 CACTTTCCTGTGAGCCAG 82 146 25341 3′ UTR 948 AGACACTTTCCTGTGAGC 69 147 25342 3′ UTR 966 ACTCTGGGTCCCTACTGC 20 148 25343 3′ UTR 992 TGCAGAAACAACTCCAGG 0 149

[0142] As shown in Table 19, SEQ ID NOs 113, 114, 115, 122, 123, 124, 125, 126, 127, 129, 131, 132, 133, 134, 135, 144, 145, 146 and 147 demonstrated at least 50% inhibition of RhoC expression in this assay and are therefore preferred.

Example 17 Antisense Inhibition of RhoC Expression-Phosphorothioate 2′-MOE Gapmer Oligonucleotides

[0143] In accordance with the present invention, a second series of oligonucleotides targeted to human RhoC were synthesized. The oligonucleotide sequences are shown in Table 20. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. L25081), to which the oligonucleotide binds.

[0144] All compounds in Table 20 are chimeric oligonucleotides (“gapmers”) 18 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by four-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. Cytidine residues in the 2′-MOE wings are 5-methylcytidines. TABLE 20 Nucleotide Sequences of Human RhoC Gapmer Oligonucleotides NUCLEOTIDE TARGET GENE GENE ISIS SEQUENCE SEQ ID NUCLEOTIDE TARGET NO. (5′→3′) NO: CO-ORDINATES¹ REGION 25344 GAGCTGAGATGAAGTCAA 110 0004-0021 5′-UTR 25345 GCTGAAGTTCCCAGGCTG 111 0044-0061 5′-UTR 25346 CCGGCTGAAGTTCCCAGG 112 0047-0064 5′-UTR 25347 GGCACCATCCCCAACGAT 113 0104-0121 Coding 25348 AGGCACCATCCCCAACGA 114 0105-0122 Coding 25349 TCCCACAGGCACCATCCC 115 0111-0128 Coding 25350 AGGTCTTCCCACAGGCAC 116 0117-0134 Coding 25351 ATGAGGAGGCAGGTCTTC 117 0127-0144 Coding 25352 TTGCTGAAGACGATGAGG 118 0139-0156 Coding 25353 TCAAAGACAGTAGGGACG 119 0178-0195 Coding 25354 TTCTCAAAGACAGTAGGG 120 0181-0198 Coding 25355 AGTTCTCAAAGACAGTAG 121 0183-0200 Coding 25356 TGTTTTCCAGGCTGTCAG 122 0342-0359 Coding 25357 TCGTCTTGCCTCAGGTCC 123 0433-0450 Coding 25358 GTGTCCTCGTCTTGCCTC 124 0439-0456 Coding 25359 CTCCTGGTGTGCTCGTCT 125 0445-0462 Coding 25360 CAGACCGAACGGCCTCCT 126 0483-0500 Coding 25361 TTCCTCAGACCGAACGGG 127 0488-0505 Coding 25362 ACTCAAGGTAGCCAAAGG 128 0534-0551 Coding 25363 CTCCCGCACTCCCTCCTT 129 0566-0583 Coding 25364 CTCAAACACCTCCCGCAC 130 0575-0592 Coding 25365 GGCCATCTCAAACACCTC 131 0581-0598 Coding 25366 CTTGTTCTTGCGGACCTG 132 0614-0631 Coding 25367 CCCCTCCGACGCTTGTTC 133 0625-0642 Coding 25368 GTATGGAGCCCTCAGGAG 134 0737-0754 3′-UTR 25369 GAGCCTTCAGTATGGAGC 135 0746-0763 3′-UTR 25370 GAAAATGGAGCCTTCAGT 136 0753-0770 3′-UTR 25371 GGAACTGAAAATGGAGCC 137 0759-0776 3′-UTR 25372 GGAGGGAACTGAAAATGG 138 0763-0780 3′-UTR 25373 GCAGGAGGGAACTGAAAA 139 0766-0783 3′-UTR 25374 AGGGCAGGGCATAGGCGT 140 0851-0868 3′-UTR 25375 GGAAGGGCAGGGCATAGG 141 0854-0871 3′-UTR 25376 CATGAGGAAGGGCAGGGC 142 0859-0876 3′-UTR 25377 TAAAGTGCTGGTGTGTGA 143 0920-0937 3′-UTR 25378 CCTGTGAGCCAGAAGTGT 144 0939-0956 3′-UTR 25379 TTCCTGTGAGCCAGAAGT 145 0941-0958 3′-UTR 25380 CACTTTCCTGTGAGCCAG 146 0945-0962 3′-UTR 25381 AGACACTTTCCTGTGAGC 147 0948-0965 3′-UTR 25382 ACTCTGGGTCCCTACTGC 148 0966-0983 3′-UTR 25383 TGCAGAAACAACTCCAGG 149 0992-1009 3′-UTR

[0145] RhoC inhibition data for these compounds were obtained by real-time quantitative PCR as described in other examples herein and are averaged from three experiments. Data are shown in Table 21. If present, “N.D.” indicates “no data”. TABLE 21 Inhibition of RhoC mRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap % TARGET Inhi- SEQ ID ISIS# REGION SITE SEQUENCE bition NO. 25344 5′ UTR 4 GAGCTGAGATGAAGTCAA 0 110 25345 5′ UTR 44 GCTGAAGTTCCCAGGCTG 35 111 25346 5′ UTR 47 CCGGCTGAAGTTCCCAGG 53 112 25347 Coding 104 GGCACCATCCCCAACGAT 50 113 25348 Coding 105 AGGCACCATCCCCAACGA 56 114 25349 Coding 111 TCCCACAGGCACCATCCC 4 115 25350 Coding 117 AGGTCTTCCCACAGGCAC 11 116 25351 Coding 127 ATGAGGAGGCAGGTCTTC 6 117 25352 Coding 139 TTGCTGAAGACGATGAGG 15 118 25353 Coding 178 TCAAAGACAGTAGGGACG 32 119 25354 Coding 181 TTCTCAAAGACAGTAGGG 7 120 25355 Coding 183 AGTTCTCAAAGACAGTAG 39 121 25356 Coding 342 TGTTTTCCAGGCTGTCAG 59 122 25357 Coding 433 TCGTCTTGCCTCAGGTCC 48 123 25358 Coding 439 GTGTGCTCGTCTTGCCTC 36 124 25359 Coding 445 CTCCTCGTGTGCTCGTCT 61 125 25360 Coding 483 CAGACCGAACGGGCTCCT 50 126 25361 Coding 488 TTCCTCAGACCGAACGGG 14 127 25362 Coding 534 ACTCAAGGTAGCCAAAGG 32 128 25363 Coding 566 CTCCCGCACTCCCTCCTT 21 129 25364 Coding 575 CTCAAACACCTCCCGCAC 9 130 25365 Coding 581 GGCCATCTCAAACACCTC 66 131 25366 Coding 614 CTTGTTCTTGCGGACCTG 61 132 25367 Coding 625 CCCCTCCGACGCTTGTTC 0 133 25368 3′ UTR 737 GTATGGAGCCCTCAGGAG 28 134 25369 3′ UTR 746 GAGCCTTCAGTATGGAGC 32 135 25370 3′ UTR 753 GAAAATGGAGCCTTCAGT 0 136 25371 3′ UTR 759 GGAACTGAAAATGGAGCC 40 137 25372 3′ UTR 763 GGAGGGAACTGAAAATGG 45 133 25373 3′ UTR 766 GCAGGAGGGAACTGAAAA 35 139 25374 3′ UTR 351 AGGGCAGGGCATAGGCGT 5 140 25375 3′ UTR 854 GGAAGGGCAGGGCATAGG 0 141 25376 3′ UTR 859 CATGAGGAAGGGCAGGGC 0 142 25377 3′ UTR 920 TAAAGTGCTGGTGTGTGA 20 143 25378 3′ UTR 939 CCTGTGAGCCAGAAGTGT 67 144 25379 3′ UTR 941 TTCCTGTGAGCCAGAAGT 61 145 25380 3′ UTR 945 CACTTTCCTGTGAGCCAG 80 146 25381 3′ UTR 943 AGACACTTTCCTGTGAGC 0 147 25382 3′ UTR 966 ACTCTGGGTCCCTACTGC 0 148 25383 3′ UTR 992 TGCAGAAACAACTCCAGG 0 149

[0146] As shown in Table 21, SEQ ID NOs 111, 112, 113, 114, 119, 121, 122, 123, 124, 125, 126, 128, 131, 132, 134, 135, 137, 138, 139, 144, 145 and 146 demonstrated at least 25% inhibition of RhoC expression in this experiment and are therefore preferred.

Example 18 Synthesis of RhoG Antisense Oligonucleotide Sequences

[0147] Oligonucleotide sequences designed to target human RhoG were synthesized as described in previous examples and are shown in Table 22. Target sequence data are from the RhoG cDNA sequence published by Vincent, S., et al. (Mol. Cell. Biol. 1992, 12, 3138-3148); Genbank accession number X61587, provided herein as SEQ ID NO: 150. TABLE 22 Nucleotide Sequences of Human RhoG Phosphorothioate Oligodeoxynucleotide NUCLEOTIDE TARGET GENE GENE ISIS SEQUENCE SEQ ID NUCLEOTIDE TARGET NO. (5′→3′) NO: CO-ORDINATES¹ REGION 25464 GACCTGGTGCCCCTCCCG 151 0048-0065 5′-UTR 25465 TCTTCTGGACCCCTCTGG 152 0073-0090 5′-UTR 25466 GGCAGTGCCTCCTCTCTC 153 0089-0106 5′-UTR 25467 GTGCAGTTGCTGTAGTGA 154 0107-0124 5′-UTR 25468 GCATCGTGGGTGCAGTTG 155 0116-0133 AUG 25469 CCACCACGCACTTGATGC 156 0137-0154 Coding 25470 TTGTGTAGCAGATGAGCA 157 0185-0202 Coding 25471 AAAGCGTTAGTTGTGTAG 158 0195-0212 Coding 25472 GCGCGCTGTAATTGTCGA 159 0239-0256 Coding 25473 GGTTCACTGTGCGCCCGT 160 0269-0286 Coding 25474 GTCCCACAGGTTCAGGTT 161 0283-0300 Coding 25475 TGTACGGAGGCGGTCATA 162 0319-0336 Coding 25476 ACGTTGGTCTGAGGGTAG 163 0342-0359 Coding 25477 CAATGGAGAAACAGATGA 164 0365-0382 Coding 25478 CATAGGACGGCGGACTGG 165 0383-0400 Coding 25479 CGCACGTTCTCATAGGAC 166 0393-0410 Coding 25480 ACCTCTGGATGCCACTTG 167 0414-0431 Coding 25481 AGGGCAGTGGTGGCACAC 168 0430-0447 Coding 25482 CAGCAGGATGGCCACATC 169 0448-0465 Coding 25483 GGGTGTCAGGCTGGGCTC 170 0488-0505 Coding 25484 CCCTGCTGCGGTGTGATG 171 0537-0554 Coding 25485 CGCGAGTGCCTGGCCCTG 172 0550-0567 Coding 25486 GTAGCGCACAGCGTGGAT 173 0574-0591 Coding 25487 CATTCGAGGTAGCGCACA 174 0582-0599 Coding 25488 ACACCATCCTGTTGCAGG 175 0606-0623 Coding 25489 GAACACTTCCTTGACACC 176 0619-0636 Coding 25490 ACAGCCTCGGCGAACACT 177 0630-0647 Coding 25491 AAGAGGATGCAGGACCGC 178 0684-0701 Coding 25492 GCAGCCTCCAAGCCAAGT 179 0713-0730 3′-UTR 25493 AAAAGGCATTCAGGGAAC 180 0818-0835 3′-UTR 25494 GGGTCCAACCTTGGCTTG 181 0936-0953 3′-UTR 25495 GTCAGTAGCGGAAAATGG 182 0984-1001 3′-UTR 25496 AGCTGGATGAACTGGTCA 183 0998-1015 3′-UTR 25497 AACTGTGTGGAAAGCTGG 184 1010-1027 3′-UTR 25498 ACCACAATAGGCAGCAAC 185 1028-1045 3′-UTR 25499 GAGGGCAGAGGTTAGAGA 186 1074-1091 3′-UTR 25500 CAATTCCAAGAGCAGCGA 187 1090-1107 3′-UTR 25501 TGGAGAAGGGAGAGAGCA 188 1119-1136 3′-UTR 25502 ACATTCACCTTCTCAGGA 189 1154-1171 3′-UTR 25503 GTCAGCAAATGCGTAAGG 190 1199-1216 3′-UTR

[0148] The compounds in Table 22 were analyzed for effect on RhoG mRNA levels by quantitative real-time PCR as described in other examples herein. Data, shown in Table 23, are averages from three experiments. If present, “N.D.” indicates “no data”. TABLE 23 Inhibition of RhoG mRNA levels by phosphorothioate oligodeoxynucleotides % TARGET Inhi- SEQ ID ISIS# REGION SITE SEQUENCE bition NO. 25464 5′ UTR 48 GACCTGGTGCCCCTCCCG 35 151 25465 5′ UTR 73 TCTTCTGGACCCCTCTGG 36 152 25466 5′ UTR 89 GGCAGTGCCTCCTCTCTC 35 153 25467 5′ UTR 107 GTGCAGTTGCTGTAGTGA 10 154 25468 5′ UTR 116 GCATCGTGGGTGCAGTTG 47 155 25469 CODING 137 CCACCACGCACTTGATGC 14 156 25470 CODING 185 TTGTGTAGCAGATGAGCA 35 157 25471 CODING 195 AAAGCGTTAGTTGTGTAG 0 158 25472 CODING 239 GCGCGCTGTAATTGTCGA 36 159 25473 CODING 269 GGTTCACTGTGCGCCCGT 16 160 25474 CODING 283 GTCCCACAGGTTCAGGTT 31 161 25475 CODING 319 TGTACGGAGGCGGTCATA 37 162 25476 CODING 342 ACGTTGGTCTGAGGGTAG 38 163 25477 CODING 365 CAATGGAGAAACAGATGA 0 164 25478 CODING 383 CATAGGACGGCGGACTGG 17 165 25479 CODING 393 CGCACGTTCTCATAGGAC 24 166 25480 CODING 414 ACCTCTGGATGCCACTTG 35 167 25481 CODING 430 AGGGCAGTGGTGGCACAC 15 168 25482 CODING 448 CAGCAGGATGGGCACATC 20 169 25483 CODING 488 GGGTGTCAGGCTGGGCTC 15 170 25484 CODING 537 CCCTGCTGCGGTGTGATG 44 171 25464 5′ UTR 48 GACCTGGTGCCCCTCCCG 35 151 25465 5′ UTR 73 TCTTCTGGACCCCTCTGG 36 152 25466 5′ UTR 89 GGCAGTGCCTCCTCTCTC 35 153 25485 CODING 550 CGCGAGTGCCTGGCCCTG 9 172 25486 CODING 574 GTAGCGCACAGCGTGGAT 35 173 25487 CODING 582 CATTCGAGGTAGCGCACA 39 174 25488 CODING 606 ACACCATCCTGTTGCAGG 23 175 25489 CODING 619 GAACACTTCCTTGACACC 31 176 25490 CODING 630 ACAGCCTCGGCGAACACT 6 177 25491 CODING 684 AAGAGGATGCAGGACCGC 18 178 25492 3′ UTR 713 GCAGCCTCCAAGCCAAGT 42 179 25493 3′ UTR 818 AAAAGGCATTCAGGGAAC 0 180 25494 3′ UTR 936 GGGTCCAACCTTGGCTTG 58 181 25495 3′ UTR 984 GTCAGTAGCGGAAAATGG 0 182 25496 3′ UTR 998 AGCTGGATGAACTGGTCA 23 183 25497 3′ UTR 1010 AACTGTGTGGAAAGCTGG 8 184 25498 3′ UTR 1028 ACCACAATAGGCAGCAAC 31 185 25499 3′ UTR 1074 GAGGGCAGAGGTTAGAGA 21 186 25500 3′ UTR 1090 CAATTCCAAGAGCAGCGA 18 187 25501 3′ UTR 1119 TGGAGAAGGGAGAGAGCA 32 188 25502 3′ UTR 1154 ACATTCACCTTCTCAGGA 20 189 25503 3′ UTR 1199 GTCAGCAAATGCGTAAGG 24 190

[0149] As shown in Table 23, SEQ ID NOs 151, 152, 153, 155, 157, 159, 161, 162, 163, 167, 171, 173, 174, 176, 179, 181, 185 and 188 demonstrated at least 25% inhibition of RhoG expression in this assay and are therefore preferred.

Example 19 Antisense Inhibition of RhoG Expression-Phosphorothioate 2′-MOE Gapmer Oligonucleotides

[0150] In accordance with the present invention, a second series of oligonucleotides targeted to human RhoG were synthesized. The oligonucleotide sequences are shown in Table 24. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. X61587), to which the oligonucleotide binds.

[0151] All compounds in Table 24 are chimeric oligonucleotides (“gapmers”) 18 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by four-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. Cytidine residues in the 2′-MOE wings are 5-methylcytidines. TABLE 24 Nucleotide Sequences of Human RhoG Gapmer Oligonucleotides NUCLEOTIDE TARGET GENE GENE ISIS SEQUENCE SEQ ID NUCLEOTIDE TARGET NO. (5′→3′) NO: CO-ORDINATES¹ REGION 25504 GACCTGGTGCCCCTCCCG 151 0048-0065 5′-UTR 25505 TCTTCTGGACCCCTCTGG 152 0073-0090 5′-UTR 25506 GGCAGTGCCTCCTCTCTC 153 0089-0106 5′-UTR 25507 GTGCAGTTGCTCTAGTGA 154 0107-0124 5′-UTR 25508 GCATCGTGGGTGCAGTTG 155 0116-0133 AUG 25509 CCACCACGCACTTGATGC 156 0137-0154 Coding 25510 TTGTGTAGCACATGAGCA 157 0185-0202 Coding 25511 AAAGCGTTAGTTGTGTAG 158 0195-0212 Coding 25512 GCGCGCTGTAATTGTCGA 159 0239-0256 Coding 25513 GGTTCACTGTGCGCCCGT 160 0269-0286 Coding 25514 GTCCCACAGGTTCAGGTT 161 0283-0300 Coding 25515 TGTACGGAGGCGGTCATA 162 0319-0336 Coding 25516 ACGTTGGTCTGAGGGTAG 163 0342-0359 Coding 25517 CAATGGAGAAACAGATGA 164 0365-0382 Coding 25518 CATAGGACGGCGGACTGG 165 0383-0400 Coding 25519 CGCACGTTCTCATAGGAC 166 0393-0410 Coding 25520 ACCTCTGGATGCCACTTG 167 0414-0431 Coding 25521 AGGGCAGTGGTGGCACAC 168 0430-0447 Coding 25522 CAGCAGGATGCGCACATC 169 0448-0465 Coding 25523 GGGTGTCAGGCTGGGCTC 170 0488-0505 Coding 25524 CCCTGCTGCGGTGTGATG 171 0537-0554 Coding 25525 CGCGAGTGCCTGGCCCTG 172 0550-0567 Coding 25526 GTAGCGCACAGCGTGGAT 173 0574-0591 Coding 25527 CATTCGAGGTAGCGCACA 174 0582-0599 Coding 25528 ACACCATCCTGTTGCAGG 175 0606-0623 Coding 25529 GAACACTTCCTTGACACC 176 0619-0636 Coding 25530 ACAGCCTCGGCGAACACT 177 0630-0647 Coding 25531 AAGAGGATGCAGGACCGC 178 0684-0701 Coding 25532 GCAGCCTCCAAGCCAAGT 179 0713-0730 3′-UTR 25533 AAAAGGCATTCAGGGAAC 180 0818-0835 3′-UTR 25534 GGGTCCAACCTTGGCTTG 181 0936-0953 3′-UTR 25535 GTCAGTAGCGGAAAATGG 182 0984-1001 3′-UTR 25536 AGCTGGATGAACTGGTCA 183 0998-1015 3′-UTR 25537 AACTGTGTGGAAAGCTGG 184 1010-1027 3′-UTR 25538 ACCACAATAGGCAGCAAC 185 1028-1045 3′-UTR 25539 GAGGGCAGAGGTTAGAGA 186 1074-1091 3′-UTR 25540 CAATTCCAAGAGCAGCGA 187 1090-1107 3′-UTR 25541 TGGAGAAGGGAGAGAGCA 188 1119-1136 3′-UTR 25542 ACATTCACCTTCTCAGGA 189 1154-1171 3′-UTR 25543 GTCAGCAAATGCGTAAGG 190 1199-1216 3′-UTR

[0152] RhoG inhibition data for compounds in Table 24 were obtained by real-time quantitative PCR as described in other examples herein and are averaged from three experiments. Data are shown in Table 25. If present, “N.D.” indicates “no data”. TABLE 25 Inhibition of RhoG mBNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap TAR- SEQ GET % ID ISIS# REGION SITE SEQUENCE Inhibition NO. 25504 5′UTR 48 GACCTGGTGCCCCTCCCG 0 151 25505 5′UTR 73 TCTTCTGGACCCCTCTGG 32 152 25506 5′UTR 89 GGCAGTGCCTCCTCTCTC 28 153 25507 5′UTR 107 GTGCAGTTGCTGTAGTGA 0 154 25508 5′UTR 116 GCATCGTGGGTGCAGTTG 12 155 25509 Coding 137 CCACCACGCACTTGATGC 0 156 25510 Coding 185 TTGTGTAGCAGATGAGCA 0 157 25511 Coding 195 AAAGCGTTAGTTGTGTAG 33 158 25512 Coding 239 GCGCGCTGTAATTGTCGA 0 159 25513 Coding 269 GGTTCACTGTCCGCCCGT 82 160 25514 Coding 283 GTCCCACAGGTTCAGGTT 0 161 25515 Coding 319 TGTACGCACGCCGTCATA 13 162 25516 Coding 342 ACGTTGGTCTCAGGGTAG 53 163 25517 Coding 365 CAATGGAGAAACAGATGA 0 164 25518 Coding 383 CATAGGACGGCGGACTGG 55 165 25519 Coding 393 CGCACGTTCTCATAGGAC 9 166 25520 Coding 414 ACCTCTCGATGCCACTTG 56 167 25521 Coding 430 AGGGCAGTGGTGGCACAC 0 168 25522 Coding 448 CAGCAGGATGGGCACATC 0 169 25523 Coding 488 GGGTCTCAGGCTGGGCTC 27 170 25524 Coding 537 CCCTGCTGCGGTGTGATG 55 171 25525 Coding 550 CGCGAGTGCCTGGCCCTG 41 172 25526 Coding 574 GTAGCGCACAGCGTGGAT 41 173 25527 Coding 582 CATTCGAGGTAGCGCACA 0 174 25528 Coding 606 ACACCATCCTCTTGCAGG 37 175 25529 Coding 619 GAACACTTCCTTCACACC 23 176 25530 Coding 630 ACAGCCTCGGCGAACACT 59 177 25531 Coding 684 AAGAGGATGCAGGACCGC 39 178 25532 3′UTR 713 GCAGCCTCCAAGCCAAGT 13 179 25533 3′UTR 818 AAAAGGCATTCAGGGAAC 43 180 25534 3′UTR 936 GGGTCCAACCTTGGCTTG 78 181 25535 3′UTR 984 GTCAGTAGCGGAAAATGG 54 182 25536 3′UTR 998 AGCTGGATGAACTGGTCA 54 183 25537 3′UTR 1010 AACTCTGTGCAAAGCTGG 59 184 25538 3′UTR 1028 ACCACAATAGGCAGCAAC 43 185 25539 3′UTR 1074 GAGGGCAGAGGTTAGAGA 0 186 25540 3′UTR 1090 CAATTCCAAGAGCAGCGA 26 187 25541 3′UTR 1119 TGGAGAACGGAGAGAGCA 0 188 25542 3′UTR 1154 ACATTCACCTTCTCAGGA 26 189 25543 3′UTR 1199 GTCACCAAATGCGTAAGG 73 190

[0153] As shown in Table 25, SEQ ID NOs 152, 158, 160, 163, 165, 167, 171, 172, 173, 175, 177, 178, 180, 181, 182, 183, 184, 185 and 190 demonstrated at least 30% inhibition of RhoG expression in this experiment and are therefore preferred.

Example 20 Human Rac1 Oligonucleotide Sequences

[0154] Antisense oligonucleotides were designed to target human Rac1. Target sequence data are from the Rac1 cDNA sequence published by Didsbury, J., et al. (J. Biol. Chem. 1989, 264, 16378-16382); Genbank accession number M29870, provided herein as SEQ ID NO: 191. Oligonucleotides were synthesized primarily with phosphorothioate linkages. Oligonucleotide sequences are shown in Table 26.

[0155] Cells were cultured, treated with oligonucleotides, and mRNA was isolated and quantitated as described in Example 2. A 45-mer antisense oligonucleotide to Rac1 (5′-ATAGAATGTGAGTCTGAACTCTTACATTTAGAACAAACAAAACCT-3′ SEQ ID NO. 192) was used as a probe as described in Didsbury, J., et al. (J. Biol. Chem. 1989, 264, 16378-16382).

[0156] An initial screen of Rac1 specific antisense oligonucleotides was performed using a oligonucleotide concentration of 300 nM.

[0157] Results are shown in Table 27. Oligonucleotides 16052 (SEQ ID NO. 195), 16056 (SEQ ID NO. 199), 16058 (SEQ ID NO. 201), 16062 (SEQ ID NO. 204) and 16063 (SEQ ID NO. 205) gave better than 50% inhibition of Rac1 mRNA levels. Oligonucleotides 16052 (SEQ ID NO. 195), 16058 (SEQ ID NO. 201) and 16062 (SEQ ID NO. 204) gave better than 75% inhibition. TABLE 26 Nucleotide Sequences of Rac-1 Phosphorothioate Oligonucleotides TARGET GENE GENE NUCLEOTIDE SEQUENCE NUCLEOTIDE TARGET ISIS NO. (5′ ->3′) SEQ ID NO: CO-ORDINATES¹ REGION 16050 CAAATGATGCAGGACTCACA 193 0252-0271 Coding 16051 CACCACCACACACTTGATC 194 0009-0027 Coding 16052 ATAAGCCCAGATTCACCG 195 0149-0166 Coding 16053 TCTTTGCGGATAGGATAGG 196 0207-0225 Coding 16054 GCTTCTTCTCCTTCAGTTTCTC 197 0379-0400 Coding 16055 CAGCACCAATCTCCTTAGC 198 0436-0454 Coding 16056 CTCTTCCTCTTCTTCACCC 199 0542-0560 Coding 16057 CCTAAGATCAAGTTTAGTTC 200 0341-0360 Coding 16058 CGCACCTCAGGATACCACTT 201 0286-0305 Coding 16059 ATCTACCATAACATTGGCAG 202 0122-0141 Coding 16060 TAATTGTCAAAGACAGTAGG 203 0100-0119 Coding 16062 GAGCGCCGAGCACTCCAGGT 204 0461-0480 Coding 16063 CTCAAACACTGTCTTGAGGC 205 0491-0510 Coding 16143 ATAGAATGTGAGTCTCAACT 206 unknown³ 3′-UTR 16144 CTTACATTTAGAACAAACAAAACCT 207 unknown³ 3′-UTR 16849 CCCAGCTAAGAATTCCGCTC 208 16058 control 16850 TAAACGCCGAATCTACGC 209 16052 control

[0158] TABLE 27 Activities of Phosphorothioate Oligonucleotides Targeted to Human Rac1 SEQ GENE ISIS ID TARGET % mRNA % mRNA No: NO: REGION EXPRESSION INHIBITION LIPOFECTIN — — 100.0% 0.0% only 16051 194 Coding 77.1% 22.9% 16052 195 Coding 3.7% 96.3% 16053 196 Coding 68.4% 31.6% 16054 197 Coding 67.6% 32.4% 16055 198 Coding 70.8% 29.2% 16056 199 Coding 48.0% 52.0% 16057 200 Coding 97.3% 2.7% 16058 201 Coding 22.2% 77.8% 16059 202 Coding 57.7% 42.3% 16060 203 Coding 91.6% 8.4% 16062 204 Coding 21.7% 78.3% 16063 205 Coding 32.4% 67.6% 16143 206 3′-UTR 56.1% 43.9% 16144 207 3′-UTR 72.9% 27.1%

Example 21 Dose Response and Specificity of Antisense Oligonucleotide Effects on Human Rac1 mRNA Levels in A549 Cells

[0159] Oligonucleotides 16050 (SEQ ID NO. 193), 16052 (SEQ ID NO. 195)16058 (SEQ ID NO. 201), 16062 (SEQ ID NO. 204) and 16143 (SEQ ID NO. 206) were chosen for dose response studies. Oligonucleotide 16057 (SEQ ID NO. 200) was chosen as a negative control because it was inactive in the initial screen. Results are shown in Table 28. Oligonucleotides 16050, 16052, 16058 and 16062 inhibited Rac1 mRNA expression in a dose dependent manner with maximum expression of 65% to 82%.

[0160] The specificity of oligonucleotides 16052 and 16058 was tested using scrambled controls. Results are shown in Table 29. Both sequences inhibited Rac1 mRNA expression in a dose dependent manner and were significantly better than their scrambled controls. TABLE 28 Dose Response of A549 Cells to Rac1 Antisense Oligonucleotides (ASOs) SEQ ID ASO Gene % mRNA % mRNA ISIS # NO: Target Dose Expression Inhibition control — LIPOFECTIN —  100%   0% only 16050 193 coding  75 nM 71.1% 28.9% 16050 193 ″ 150 nM 53.6% 46.4% 16050 193 ″ 300 nM 33.6% 66.4% 16052 195 coding  75 nM 68.2% 31.8% 16052 195 ″ 150 nM 40.5% 59.5% 16052 195 ″ 300 nM 28.3% 71.7% 16057 200 coding  75 nM 81.7% 18.3% 16057 200 ″ 150 nM 80.2% 19.8% 16057 200 ″ 300 nM 85.8% 14.2% 16058 201 coding  75 nM 60.1% 39.9% 16058 201 ″ 150 nM 42.9% 57.1% 16058 201 ″ 300 nM 17.7% 82.3% 16062 204 coding  75 nM 50.5% 49.5% 16062 204 ″ 150 nM 40.2% 59.8% 16062 204 ″ 300 nM 25.2% 74.8% 16143 206 3′-UTR  75 nM 294.8%  — 16143 206 ″ 150 nM 100.8%  — 16143 206 ″ 300 nM 88.6% 11.4%

[0161] TABLE 29 Specificity of Rac1 Antisense Oligonucleotides (ASOs) in A549 Cells SEQ ID ASO Gene % mRNA % mRNA ISIS # NO: Target Dose Expression Inhibition control — LIPOFECTIN —  100%   0% only 16052 195 coding  75 nM 86.6% 13.4% 16052 195 ″ 150 nM 52.8% 47.2% 16052 195 ″ 300 nM 18.5% 81.5% 16850 209 control  75 nM 88.9% 11.1% 16850 209 ″ 150 nM 97.2%  2.8% 16850 209 ″ 300 nM 107.4%  — 16058 201 coding  75 nM 82.7% 17.3% 16058 201 ″ 150 nM 36.8% 63.2% 16058 201 ″ 300 nM 21.1% 78.9% 16849 208 control  75 nN 90.7%  9.3% 16849 208 ″ 150 nM 70.2% 29.8% 16849 208 ″ 300 nM 68.2% 31.8%

Example 22 Design and Testing of Chimeric (Deoxy Gapped) 2′-O-Methoxyethyl Rac1 Antisense Oligonucleotides on Rac1 mRNA Levels in A549 Cells

[0162] Oligonucleotides targeted to Rac1 were synthesized as a uniformly phosphorothioate or mixed phosphorothioate/phosphodiester chimeric oligonucleotides having variable regions of 2′-O-methoxyethyl (2′-MOE) nucleotides and deoxynucleotides. All 2′-MOE cytosines were 5-methyl-cytosines. Additionally, some oligonucleotides were synthesized with deoxycytosines as 5-methyl-cytosines. Additional oligonucleotides were synthesized, with similar chemistries, as scrambled controls. Oligonucleotide sequences and chemistries are shown in Tables 30 and 31. A dose response experiment was performed using a number of these oligonucleotides as described in Example 3.

[0163] Results are shown in Table 32. All of the chimeric oligonucleotides tested showed a dose dependent effect and showed inhibition of Rac mRNA levels comparable to that of the phosphorothioate oligodeoxynucleotide. TABLE 30 Nucleotide Sequences of Rac1 Gapmer Oligonucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET NO. (5′ -> 3′) NO: CO-ORDINATES¹ REGION 16899 ATAAGCCCAGATTCACCG 195 0149-0166 Coding 16900 CAAATGATGCAGGACTCACA 193 0252-0271 Coding 16901 CGCACCTCAGGATACCACTT 201 0286-0305 Coding 17161 ATAAGCCCAGATTCACCG 195 0149-0166 Coding 17162 ATAAGCCCAGATTCACCG 195 0149-0166 Coding 17163 ATAAGCCCAGATTCACCG 195 0149-0166 Coding 17164 ATAAGCCCAGATTCACCG 195 0149-0166 Coding 18540 ATAAGCCCTGATTCACCG 210 16899 mismatch 18541 ATACGCCCTGATTCACCG 211 16899 mismatch 18542 ATACGCCCTGATTAACCG 212 16899 mismatch 18549 TAAACGCCGAATCTACGC 213 16899 control

[0164] TABLE 31 Nucleotide Sequences of Rac1 Mixed Backbone Oligonucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET NO. (5′ ->3′) NO: CO-ORDINATES¹ REGION 17814 ToAoAoAoCoGoCoCoGsAsAsTsCsTsAsCsGsC 213 16899 control 17815 AoToAoAoGoCoCoCoAsGsAsTsTsCsAsCsCsC 195 0149-0166 Coding 17816 CoAoAoAoToGsAsTsGsCsAsGsGsAsCsToCoAoCoA 193 0252-0271 Coding 17817 AoAoAoCoToGsCsTsGsAsAsGsTsAsCsGoCoAoCoA 214 17816 control 24686 ToAoAoAoCoGoCoCoGoAoAoToCoToAoCoGoC 213 16899 control 24687 TsAsAsAsCsGsCsCsGsAsAsTsCsTsAsCsGsC 213 16899 control

[0165] TABLE 32 Dose Response of A549 Cells to Rac1 Antisense Gapmer Oligonucleotides (ASOs) SEQ ID ASO Gene % mRNA % mRNA ISIS # NO: Target Dose Expression Inhibition control — LIPOFECTIN —  100 0.0% only 16899 195 coding  75 nM 79.9% 20.1% ″ ″ ″ 150 nM 40.8% 59.2% ″ ″ ″ 300 nM 21.8% 78.2% 17161 195 coding  75 nM 31.3% 68.7% ″ ″ ″ 150 nM 16.9% 83.1% ″ ″ ″ 300 nM 12.3% 87.7% 17162 195 coding  75 nM 89.2% 10.8% ″ ″ ″ 150 nM 63.0% 37.0% ″ ″ ″ 300 nM 18.4% 81.6% 17163 195 coding  75 nM 93.4% 6.6% ″ ″ ″ 150 nM 67.3% 32.7% ″ ″ ″ 300 nM 34.4% 65.6% 17164 195 coding  75 nM 94.7% 5.3% ″ ″ ″ 150 nM 65.9% 34.1% ″ ″ ″ 300 nM 36.2% 63.8%

Example 23 Human cdc42 Chimeric (Deoxy Gapped) 2′-O-methoxyethyl Oligonucleotide Sequences

[0166] Antisense oligonucleotides were designed to target human cdc42. Target sequence data are from the cdc42 cDNA sequence published by Shinjo, K. et al. (Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 9853-9857); Genbank accession number M57298, provided herein as SEQ ID NO: 215. Oligonucleotides were synthesized as uniformly phosphorothioate chimeric oligonucleotides having a centered deoxy gap of eight nucleotides flanked by 2′-O-methoxyethyl (2′-MOE) regions. All 2′-MOE cytosines were 5-methyl-cytosines. Oligonucleotide sequences are shown in Table 33.

[0167] A549 cells were cultured and treated with oligonucleotide as described in Example 2. Quantitation of cdc42 mRNA levels was determined by real-time PCR (RT-PCR) as described in previous examples.

[0168] For cdc42 the PCR primers were: For cdc42 the PCR primers were: Forward: 5′-TTCAGCAATGCACACAATTAAGTGT-3′ SEQ ID NO. 216 Reverse: 5′-TGTTGTGTAGGATATCAGGAGACATGT-3′ SEQ ID NO. 217 and the PCR probe was: FAM-TTGTGGGCGATGGTGCTGTTGGTA-TAMRA

[0169] (SEQ ID NO. 218) where FAM or JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

[0170] For GAPDH the PCR primers were: For GAPDH the PCR primers were: Forward primer: 5′-GAAGGTGAAGGTCGGAGTC-3′ SEQ ID NO. 65 Reverse primer: 5′-GAAGATGGTGATGGGATTTC-3′ SEQ ID NO. 66 and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′

[0171] (SEQ ID NO. 67) where FAM or JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

[0172] Results are shown in Table 34. All oligonucleotides tested gave greater than 40% inhibition of cdc42 mRNA expression. TABLE 33 Nucleotide Sequences of cdc42 Oligonucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET NO. (5′ ->3′) NO: CO-ORDINATES¹ REGION 17203 TAATTGTCTGCATTGCTGAA 219 0063-0082 AUG 17209 TTACCAACAGCACGATCGCC 220 0097-0116 Coding 17210 CCACCAATCATAACTGTGAC 221 0193-0212 Coding 17211 GTGGATAACTCAGCGGTCGT 222 0270-0289 Coding 17212 GAAGATGGAGAGACCACTGA 223 0316-0335 Coding 17213 GTGAGTTATCTCAGGCACCC 224 0359-0378 Coding 17214 GCTTCTGTTTGTTCTTGGCA 225 0456-0475 Coding 17215 TGACAGCCTTCAGGTCACGG 226 0507-0526 Coding 17216 CACCTGCGGCTCTTCTTCGG 227 0613-0632 Coding 17217 TTGTCTCACACGAGTGCATG 228 0774-0793 3′-UTR 17218 TTCTGACAATACAATTACTC 229 0844-0863 3′-UTR 17219 TTACAGAGTCATCCACAAGC 230 0961-0980 3′-UTR 20457 CGATAGTCTCCACGTGAGGC 231 17215 control 21668 CGATAGTCTCCACGTGAGGC 231 17215 control 21917 GTAACGCTCCTATGGCCAGG 232 17215 control 21918 AGACTGACTGCTCGTCGCGA 233 17215 control

[0173] TABLE 34 Activities of Phosphorothioate Oligonucleotides Targeted to Human Cdc42 SEQ GENE ISIS ID TARGET % mRNA 5 mRNA No: NO: REGION EXPRESSION INHIBITION LIPOFECTIN — —  100%   0% only 17208 219 AUG 40.6% 59.4% 17209 220 Coding 43.4% 56.6% 17210 221 Coding 55.4% 44.6% 17211 222 Coding 25.5% 74.5% 17212 223 Coding 31.1% 68.9% 17213 224 Coding 14.0% 86.0% 17214 225 Coding 27.4% 72.6% 17215 226 Coding 16.9% 83.1% 17216 227 Coding 26.0% 74.0% 17217 228 3′-UTR 28.4% 71.6% 17218 229 3′-UTR 17.2% 82.8% 17219 230 3′-UTR 20.2% 79.8%

Example 24 Dose Response of Antisense Oligonucleotide Effects on Human cdc42 mRNA Levels in A549 Cells

[0174] Oligonucleotides 17213 (SEQ ID NO. 224), 17215 (SEQ ID NO. 226), 17218 (SEQ ID NO. 229), and 17219 (SEQ ID NO. 230) were chosen for dose response studies. Results are shown in Table 35. TABLE 35 Dose Response of A549 Cells to Cdc42 Antisense Oligonucleotides (ASOs) SEQ ID ASO Gene % mRNA % mRNA ISIS # NO: Target Dose Expression Inhibition control — LIPOFECTIN — 100% 0% only 17213 224 coding  75 nM 158% — 17213 ″ ″ 300 nM 16% 84% 17215 226 coding  75 nM 90% 10% 17215 ″ ″ 300 nM 21% 79% 17218 229 3′-UTR  75 nM 53% 47% 17218 ″ ″ 300 nM 38% 62% 17219 230 3′-UTR  75 nM 102% — 17219 ″ ″ 300 nM 41% 59%

Example 25 Additional cdc42 Chimeric Oligonucleotides

[0175] Oligonucleotides having SEQ ID NO: 226 were synthesized as mixed phosphorothioate/phosphodiester chimeric oligonucleotides having variable wing regions of 2′-O-methoxyethyl (2′-MOE) nucleotides and a central stretch of nine deoxynucleotides. All 2′-MOE cytosines were 5-methyl-cytosines. Oligonucleotide sequences and chemistries are shown in Table 36. TABLE 36 Nucleotide Sequence of 17215 Analog SEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET NO. (5′ ->3′) NO: CO-ORDINATES¹ REGION 22276 ToGoAoCoAoGsCsCsTsTsCsAsGsGsTsCoAoCoGoG 226 0507-0526 Coding 22277 CoGoAoToAoGsTsCsTsCsCsAsCsGsTsGoAoGoGoC 231 22276 control

[0176]

1 233 1 1074 DNA Homo sapiens 1 gaattcgggc taccctcgcc ccgcccgcgg tcctccgtcg gttctctcat tagtccacgg 60 tctggtcttc agctacccgc cttcgtctcc gagtttgcga ctcgcgggac cggcgtcccc 120 ggcgcgaaga ggctggactc ggattcgttg cctgagcaat ggctgccatc cggaagaaac 180 tggtgattgt tggtgatgga gcctgtggaa agacatgctt gctcatagtc ttcagcaagg 240 accagttccc agaggtgtat gtgcccacag tgtttgagaa ctatgtggca gatatcgagg 300 tggatggaaa gcaggtagag ttggctttgt gggacacagc tgggcaggaa gattatgatc 360 gcctgaggcc cctctcctac ccagataccg atgttatact gatgtgtttt tccatcgaca 420 gccctgatag tttagaaaac atcccagaaa agtggacccc agaagtcaag catttctgtc 480 ccaacgtgcc catcatcctg gttgggaata agaaggatct tcggaatgat gagcacacaa 540 ggcgggagct agccaagatg aagcaggagc cggtgaaacc tgaagaaggc agagatatgg 600 caaacaggat tggcgctttt gggtacatgg agtgttcagc aaagaccaaa gatggagtga 660 gagaggtttt tgaaatggct acgagagctg ctctgcaagc tagacgtggg aagaaaaaat 720 ctggttgcct tgtcttgtga aaccttgctg caagcacagc ccttatgcgg ttaattttga 780 agtgctgttt attaatctta gtgtatgatt actggccttt ttcatttatc tataatttac 840 ctaagattac aaatcagaag tcatcttgct accagtattt agaagccaac tatgattatt 900 aacgatgtcc aacccgtctg gcccaccagg gtccttttga cactgctcta acagccctcc 960 tctgcactcc cacctgacac accaggcgct aattcaagga atttcttaac ttcttgcttc 1020 tttctagaaa gagaaacagt tggtaacttt tgtcaattag gctgtaacta cttt 1074 2 19 DNA Artificial Sequence Description of Artificial SequenceSynthetic 2 tgcaagcaca gcccttatg 19 3 19 DNA Artificial Sequence Description of Artificial SequenceSynthetic 3 tgtcaaaagg accctggtg 19 4 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 4 agtcgcaaac tcggagac 18 5 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 5 ttgctcaggc aacgaatc 18 6 22 DNA Artificial Sequence Description of Artificial SequenceSynthetic 6 ctgaagacta tgagcaagca tg 22 7 19 DNA Artificial Sequence Description of Artificial SequenceSynthetic 7 ctcatcattc cgaagatcc 19 8 22 DNA Artificial Sequence Description of Artificial SequenceSynthetic 8 ccaatcctgt ttgccatatc tc 22 9 22 DNA Artificial Sequence Description of Artificial SequenceSynthetic 9 ccatctttgg tctttgctga ac 22 10 21 DNA Artificial Sequence Description of Artificial SequenceSynthetic 10 gcagagcagc tctcgtagcc a 21 11 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 11 tcacaagaca aggcaaccag 20 12 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 12 aggccagtaa tcatacacta 20 13 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 13 gttggcttct aaatactggt 20 14 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 14 ggctgttaga gcagtgtcaa 20 15 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 15 agcgcctggt gtgtcaggtg 20 16 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 16 tagttacagc ctaattgaca 20 17 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 17 ggcacctgtt gggtgagctg 20 18 22 DNA Artificial Sequence Description of Artificial SequenceSynthetic 18 acactcttgc ttaccgtacc tt 22 19 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 19 tgcggtaagt gcggtatcaa 20 20 19 DNA Artificial Sequence Description of Artificial SequenceSynthetic 20 gtcgttagtc gaaatgagg 19 21 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 21 agcttgtgaa cgagtgtcga 20 22 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 22 tgcagttggc agagtctgaa 20 23 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 23 agagaaccga cggaggac 18 24 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 24 gtggactaat gagagaac 18 25 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 25 gaccgtggac taatgaga 18 26 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 26 agctgaagac cagaccgt 18 27 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 27 aatccgagtc cagcctct 18 28 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 28 aacgaatccg agtccagc 18 29 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 29 tcaggcaacg aatccgag 18 30 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 30 caccaacaat caccagtt 18 31 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 31 aagactatga gcaagcat 18 32 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 32 atacacctct gggaactg 18 33 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 33 acatagttct caaacact 18 34 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 34 actctacctg ctttccat 18 35 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 35 cacaaagcca actctacc 18 36 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 36 aacatcggta tctgggta 18 37 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 37 ttctgggatg ttttctaa 18 38 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 38 ggacagaaat gcttgact 18 39 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 39 gtgctcatca ttccgaag 18 40 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 40 cttgtgtgct catcattc 18 41 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 41 tagctcccgc cttgtgtg 18 42 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 42 ccaatcctgt ttgccata 18 43 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 43 gtctttgctg aacactcc 18 44 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 44 aaaacctctc tcactcca 18 45 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 45 aagacaaggc aaccagat 18 46 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 46 tttcacaaga caaggcaa 18 47 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 47 gcaaggtttc acaagaca 18 48 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 48 attaaccgca taagggct 18 49 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 49 taataaacag cacttcaa 18 50 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 50 ccagtaatca tacactaa 18 51 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 51 atgacttctg atttgtaa 18 52 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 52 tagcaagatg acttctga 18 53 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 53 ctggtagcaa gatgactt 18 54 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 54 ctaaatactg gtagcaag 18 55 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 55 ttggcttcta aatactgg 18 56 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 56 tcatagttgg cttctaaa 18 57 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 57 aataatcata gttggctt 18 58 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 58 tcaaaaggac cctggtgg 18 59 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 59 gtgcagagga gggctgtt 18 60 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 60 ccaactgttt ctctttct 18 61 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 61 aagtagttac agcctaat 18 62 19 DNA Artificial Sequence Description of Artificial SequenceSynthetic 62 ggctggactc ggattcgtt 19 63 22 DNA Artificial Sequence Description of Artificial SequenceSynthetic 63 ccatcaccaa caatcaccag tt 22 64 22 DNA Artificial Sequence Description of Artificial SequenceSynthetic 64 cctgagcaat ggctgccatc cg 22 65 19 DNA Artificial Sequence Description of Artificial SequenceSynthetic 65 gaaggtgaag gtcggagtc 19 66 20 DNA Homo sapiens Description of Artificial SequenceSynthetic 66 gaagatggtg atgggatttc 20 67 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 67 caagcttccc gttctcagcc 20 68 591 DNA Homo sapiens Description of Artificial SequenceSynthetic 68 atggcggcca tccgcaagaa gctggtggtg gtgggcgacg gcgcgtgtgg caagacgtgc 60 ctgctgatcg tgttcagtaa ggacgagttc cccgaggtgt acgtgcccac cgtcttcgag 120 aactatgtgg ccgacattga ggtggacggc aagcaggtgg agctggcgct gtgggacacg 180 gcgggccagg aggactacga ccgcctgcgg ccgctctcct acccggacac cgacgtcatt 240 ctcatgtgct tctcggtgga cagcccggac tcgctggaga acatccccga gaagtgggtc 300 cccgaggtga agcacttctg tcccaatgtg cccatcatcc tggtggccaa caaaaaagac 360 ctgcgcagcg acgagcatgt ccgcacagag ctggcccgca tgaagcagga acccgtgcgc 420 acggatgacg gccgcgccat ggccgtgcgc atccaagcct acgactacct cgagtgctct 480 gccaagacca aggaaggcgt gcgcgaggtc ttcgagacgg ccacgcgcgc cgcgctgcag 540 aagcgctacg gctcccagaa cggctgcatc aactgctgca aggtgctatg a 591 69 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 69 ccaccaccag cttcttgc 18 70 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 70 ccgtcgccca ccaccacc 18 71 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 71 gcacgtcttg ccacacgc 18 72 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 72 actgaacacg atcagcag 18 73 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 73 ttactgaaca cgatcagc 18 74 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 74 ccttactgaa cacgatca 18 75 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 75 gtccttactg aacacgat 18 76 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 76 ctcgtcctta ctgaacac 18 77 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 77 aactcgtcct tactgaac 18 78 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 78 catagttctc gaagacgg 18 79 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 79 tcggccacat agttctcg 18 80 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 80 ccgtccacct caatgtcg 18 81 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 81 aagcacatga gaatgacg 18 82 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 82 gagtccgggc tgtccacc 18 83 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 83 atgttctcca gcgagtcc 18 84 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 84 gggatgttct ccagcgag 18 85 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 85 gacatgctcg tcgctgcg 18 86 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 86 cggacatgct cgtcgctg 18 87 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 87 tgtgcggaca tgctcgtc 18 88 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 88 ctctgtgcgg acatgctc 18 89 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 89 ccagctctgt gcggacat 18 90 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 90 cgggccagct ctgtgcgg 18 91 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 91 tgcgggccag ctctgtgc 18 92 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 92 gttcctgctt catgcggg 18 93 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 93 acgggttcct gcttcatg 18 94 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 94 gtagtcgtag gcttggat 18 95 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 95 cgaggtagtc gtaggctt 18 96 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 96 gtcttggcag agcactcg 18 97 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 97 acctcgcgca cgccttcc 18 98 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 98 agacctcgcg cacgcctt 18 99 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 99 cgaagacctc gcgcacgc 18 100 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 100 ctcgaagacc tcgcgcac 18 101 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 101 gccgtctcga agacctcg 18 102 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 102 cgtggccgtc tcgaagac 18 103 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 103 gttctgggag ccgtagcg 18 104 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 104 gccgttctgg gagccgta 18 105 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 105 gatgcagccg ttctggga 18 106 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 106 gttgatgcag ccgttctg 18 107 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 107 cagcagttga tgcagccg 18 108 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 108 agcaccttgc agcagttg 18 109 1058 DNA Homo sapiens 109 gccttgactt catctcagct ccagagcccg ccctctcttc ctgcagcctg ggaacttcag 60 ccggctggag cccaccatgg ctgcaatccg aaagaagctg gtgatcgttg gggatggtgc 120 ctgtgggaag acctgcctcc tcatcgtctt cagcaaggat cagtttccgg aggtctacgt 180 ccctactgtc tttgagaact atattgcgga cattgaggtg gacggcaagc aggtggagct 240 ggctctgtgg gacgcggaca ttgaggtgga cggcaagcag gtggagctgg ctctgtggga 300 cgacactgat gtcatcctca tgtgcttctc catcgacagc cctgacagcc tggaaaacat 360 tcctgagaag tggaccccag aggtgaagca cttctgcccc aacgtgccca tcatcctggt 420 ggggaataag aaggacctga ggcaagacga gcacaccagg agagagctgg ccaagatgaa 480 gcaggagccc gttcggtctg aggaaggccg ggacatggcg aaccggatca gtgcctttgg 540 ctaccttgag tgctcagcca agaccaagga gggagtgcgg gaggtgtttg agatggccac 600 tcgggctggc ctccaggtcc gcaagaacaa gcgtcggagg ggctgtccca ttctctgaga 660 tccccccaaa gggccctttt cctacatgcc ccctcccttc acaggggtac agaaattatc 720 cccctacaac cccagcctcc tgagggctcc atactgaagg ctccattttc agttccctcc 780 tgcccaggac tgcattgttt tctagccccg aggtgtggca cgggccctcc ctcccagcgc 840 tctgggagcc acgcctatgc cctgcccttc ctcatgggcc cctggggatc ttgccccttt 900 gaccttcccc aaaggatggt cacacaccag cactttatac acttctggct cacaggaaag 960 tgtctgcagt agggacccag agtcccaggc ccctggagtt gtttctgcag gggccttgtc 1020 tctcactgca tttggtcagg ggggcatgaa taaaggct 1058 110 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 110 gagctgagat gaagtcaa 18 111 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 111 gctgaagttc ccaggctg 18 112 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 112 ccggctgaag ttcccagg 18 113 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 113 ggcaccatcc ccaacgat 18 114 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 114 aggcaccatc cccaacga 18 115 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 115 tcccacaggc accatccc 18 116 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 116 aggtcttccc acaggcac 18 117 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 117 atgaggaggc aggtcttc 18 118 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 118 ttgctgaaga cgatgagg 18 119 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 119 tcaaagacag tagggacg 18 120 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 120 ttctcaaaga cagtaggg 18 121 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 121 agttctcaaa gacagtag 18 122 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 122 tgttttccag gctgtcag 18 123 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 123 tcgtcttgcc tcaggtcc 18 124 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 124 gtgtgctcgt cttgcctc 18 125 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 125 ctcctggtgt gctcgtct 18 126 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 126 cagaccgaac gggctcct 18 127 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 127 ttcctcagac cgaacggg 18 128 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 128 actcaaggta gccaaagg 18 129 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 129 ctcccgcact ccctcctt 18 130 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 130 ctcaaacacc tcccgcac 18 131 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 131 ggccatctca aacacctc 18 132 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 132 cttgttcttg cggacctg 18 133 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 133 cccctccgac gcttgttc 18 134 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 134 gtatggagcc ctcaggag 18 135 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 135 gagccttcag tatggagc 18 136 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 136 gaaaatggag ccttcagt 18 137 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 137 ggaactgaaa atggagcc 18 138 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 138 ggagggaact gaaaatgg 18 139 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 139 gcaggaggga actgaaaa 18 140 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 140 agggcagggc ataggcgt 18 141 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 141 ggaagggcag ggcatagg 18 142 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 142 catgaggaag ggcagggc 18 143 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 143 taaagtgctg gtgtgtga 18 144 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 144 cctgtgagcc agaagtgt 18 145 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 145 ttcctgtgag ccagaagt 18 146 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 146 cactttcctg tgagccag 18 147 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 147 agacactttc ctgtgagc 18 148 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 148 actctgggtc cctactgc 18 149 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 149 tgcagaaaca actccagg 18 150 1284 DNA Homo sapiens 150 gcttctcgag cccggagccg ctgccgccgc ccccagctcc cccgcctcgg gaggggcacc 60 aggtcactgc agccagaggg gtccagaaga gagaggaggc actgcctcac tacagcaact 120 gcacccacga tgcagagcat caagtgcgtg gtggtgggtg atggggctgt gggcaagacg 180 tgcctgctca tctgctacac aactaacgct ttccccaaag agtacatccc caccgtgttc 240 gacaattaca gcgcgcagag cgcagttgac gggcgcacag tgaacctgaa cctgtgggac 300 actgcgggcc aggaggagta tgaccgcctc cgtacactct cctaccctca gaccaacgtt 360 ttcgtcatct gtttctccat tgccagtccg ccgtcctatg agaacgtgcg gcacaagtgg 420 catccagagg tgtgccacca ctgccctgat gtgcccatcc tgctggtggg caccaagaag 480 gacctgagag cccagcctga caccctacgg cgcctcaagg agcagagcca ggcgcccatc 540 acaccgcagc agggccaggc actcgcgaaa cagatccacg ctgtgcgcta cctcgaatgc 600 tcagccctgc aacaggatgg tgtcaaggaa gtgttcgccg aggctgtccg ggctgtgctc 660 aaccccacgc cgatcaagcg tgggcggtcc tgcatcctct tgtgaccctg gcacttggct 720 tggaggctgc ccctgccctc cccccaccag ttgtgccttg gtgccttgtc cgcctcagct 780 gtgccttaag gactaattct ggcacccctt tccaggggtt ccctgaatgc ctttttctct 840 gagtgccttt ttctccttaa ggaggcctgc agagaaaggg gctttgggct ctgcccctct 900 ggcttgggaa cactgggtat tctcatgagc tcatccaagc caaggttgga cccctcccca 960 agaggccaac ccagtgcccc ctcccatttt ccgctactga ccagttcatc cagctttcca 1020 cacagttgtt gctgcctatt gtggtgccgc ctcaggttag gggctctcag ccatctctaa 1080 cctctgccct cgctgctctt ggaattgcgc ccccaagatg ctctctccct tctccaatga 1140 gggagccaca gaatcctgag aaggtgaatg taccctaacc tgctcctctg tgcctaggcc 1200 ttacgcattt gctgactgac tcagccccca tgcttctggg gacctttcct acccccatca 1260 gcatcaataa aacctcctgt ctcc 1284 151 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 151 gacctggtgc ccctcccg 18 152 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 152 tcttctggac ccctctgg 18 153 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 153 ggcagtgcct cctctctc 18 154 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 154 gtgcagttgc tgtagtga 18 155 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 155 gcatcgtggg tgcagttg 18 156 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 156 ccaccacgca cttgatgc 18 157 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 157 ttgtgtagca gatgagca 18 158 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 158 aaagcgttag ttgtgtag 18 159 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 159 gcgcgctgta attgtcga 18 160 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 160 ggttcactgt gcgcccgt 18 161 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 161 gtcccacagg ttcaggtt 18 162 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 162 tgtacggagg cggtcata 18 163 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 163 acgttggtct gagggtag 18 164 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 164 caatggagaa acagatga 18 165 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 165 cataggacgg cggactgg 18 166 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 166 cgcacgttct cataggac 18 167 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 167 acctctggat gccacttg 18 168 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 168 agggcagtgg tggcacac 18 169 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 169 cagcaggatg ggcacatc 18 170 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 170 gggtgtcagg ctgggctc 18 171 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 171 ccctgctgcg gtgtgatg 18 172 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 172 cgcgagtgcc tggccctg 18 173 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 173 gtagcgcaca gcgtggat 18 174 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 174 cattcgaggt agcgcaca 18 175 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 175 acaccatcct gttgcagg 18 176 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 176 gaacacttcc ttgacacc 18 177 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 177 acagcctcgg cgaacact 18 178 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 178 aagaggatgc aggaccgc 18 179 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 179 gcagcctcca agccaagt 18 180 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 180 aaaaggcatt cagggaac 18 181 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 181 gggtccaacc ttggcttg 18 182 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 182 gtcagtagcg gaaaatgg 18 183 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 183 agctggatga actggtca 18 184 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 184 aactgtgtgg aaagctgg 18 185 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 185 accacaatag gcagcaac 18 186 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 186 gagggcagag gttagaga 18 187 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 187 caattccaag agcagcga 18 188 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 188 tggagaaggg agagagca 18 189 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 189 acattcacct tctcagga 18 190 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 190 gtcagcaaat gcgtaagg 18 191 579 DNA Artificial Sequence Description of Artificial SequenceSynthetic 191 atgcaggcca tcaagtgtgt ggtggtggga gacggagctg taggtaaaac ttgcctactg 60 atcagttaca caaccaatgc atttcctgga gaatatatcc ctactgtctt tgacaattat 120 tctgccaatg ttatggtaga tggaaaaccg gtgaatctgg gcttatggga tacagctgga 180 caagaagatt atgacagatt acgcccccta tcctatccgc aaacagatgt gttcttaatt 240 tgcttttccc ttgtgagtcc tgcatcattt gaaaatgtcc gtgcaaagtg gtatcctgag 300 gtgcggcacc actgtcccaa cactcccatc atcctagtgg gaactaaact tgatcttagg 360 gatgataaag acacgatcga gaaactgaag gagaagaagc tgactcccat cacctatccg 420 cagggtctag ccatggctaa ggagattggt gctgtaaaat acctggagtg ctcggcgctc 480 acacagcgag gcctcaagac agtgtttgac gaagcgatcc gagcagtcct ctgcccgcct 540 cccgtgaaga agaggaagag aaaatgcctg ctgttgtaa 579 192 45 DNA Artificial Sequence Description of Artificial SequenceSynthetic 192 atagaatgtg agtctgaact cttacattta gaacaaacaa aacct 45 193 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 193 caaatgatgc aggactcaca 20 194 19 DNA Artificial Sequence Description of Artificial SequenceSynthetic 194 caccaccaca cacttgatg 19 195 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 195 ataagcccag attcaccg 18 196 19 DNA Artificial Sequence Description of Artificial SequenceSynthetic 196 tgtttgcgga taggatagg 19 197 22 DNA Artificial Sequence Description of Artificial SequenceSynthetic 197 gcttcttctc cttcagtttc tc 22 198 19 DNA Artificial Sequence Description of Artificial SequenceSynthetic 198 cagcaccaat ctccttagc 19 199 19 DNA Artificial Sequence Description of Artificial SequenceSynthetic 199 ctcttcctct tcttcacgg 19 200 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 200 cctaagatca agtttagttc 20 201 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 201 cgcacctcag gataccactt 20 202 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 202 atctaccata acattggcag 20 203 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 203 taattgtcaa agacagtagg 20 204 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 204 gagcgccgag cactccaggt 20 205 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 205 gtcaaacact gtcttgaggc 20 206 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 206 atagaatgtg agtctgaact 20 207 25 DNA Artificial Sequence Description of Artificial SequenceSynthetic 207 cttacattta gaacaaacaa aacct 25 208 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 208 cccagctaag aattccgctc 20 209 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 209 taaacgccga atctacgc 18 210 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 210 ataagccctg attcaccg 18 211 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 211 atacgccctg attcaccg 18 212 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 212 atacgccctg attaaccg 18 213 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 213 taaacgccga atctacgc 18 214 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 214 aaactgctga agtacgcaca 20 215 1175 DNA Artificial Sequence Description of Artificial SequenceSynthetic 215 ccccggtgga gaagctgagg tcatcatcag atttgaaata tttaaagtgg atacaaaatt 60 atttcagcaa tgcagacaat taagtgtgtt gttgtgggcg atggtgctgt tggtaaaaca 120 tgtctcctga tatcctacac aacaaacaaa tttccatcgg aatatgtacc gactgttttt 180 gacaactatg cagtcacagt tatgattggt ggagaaccat atactcttgg actttttgat 240 actgcagggc aagaggatta tgacagatta cgaccgctga gttatccaca aacagatgta 300 tttctagtct gtttttcagt ggtctctcca tcttcatttg aaaacgtgaa agaaaagtgg 360 gtgcctgaga taactcacca ctgtccaaag actcctttct tgcttgttgg gactcaaatt 420 gatctcagag atgacccctc tactattgag aaacttgcca agaacaaaca gaagcctatc 480 actccagaga ctgctgaaaa gctggcccgt gacctgaagg ctgtcaagta tgtggagtgt 540 tctgcactta cacagaaagg cctaaagaat gtatttgacg aagcaatatt ggctgccctg 600 gagcctccag aaccgaagaa gagccgcagg tgtgtgctgc tatgaacatc tctccagagc 660 cctttctgca cagctggtgt cggcatcata ctaaaagcaa tgtttaaatc aaactaaaga 720 ttaaaaatta aaattcgttt ttgcaataat gacaaatgcc ctgcacctac ccacatgcac 780 tcgtgtgaga caaggcccat aggtatggcc ccccccttcc ccctcccagt actagttaat 840 tttgagtaat tgtattgtca gaaaagtgat tagtactatt tttttttgtt gtttcaaaaa 900 aaaaattttt gtgtgtctgt tttttttttt tttttttttt gttgtttaaa aggaaggcat 960 gcttgtggat gactctgtaa cagactaatt ggaattgttg aagctgctcc ctggttccac 1020 tctggagagt aatctgggac atcttagtgt tttgttttgt ttttttccct cctctttttt 1080 ttggggggga gtgtgtgggg ggtttgtttt ttagtcttgt ttttttaatt cattaaccag 1140 tggttaagcc cttaagggag gaggacggat tgatt 1175 216 25 DNA Artificial Sequence Description of Artificial SequenceSynthetic 216 ttcagcaatg cagacaatta agtgt 25 217 27 DNA Artificial Sequence Description of Artificial SequenceSynthetic 217 tgttgtgtag gatatcagga gacatgt 27 218 24 DNA Artificial Sequence Description of Artificial SequenceSynthetic 218 ttgtgggcga tggtgctgtt ggta 24 219 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 219 taattgtctg cattgctgaa 20 220 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 220 ttaccaacag caccatcgcc 20 221 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 221 ccaccaatca taactgtgac 20 222 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 222 gtggataact cagcggtcgt 20 223 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 223 gaagatggag agaccactga 20 224 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 224 gtgagttatc tcaggcaccc 20 225 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 225 gcttctgttt gttcttggca 20 226 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 226 tgacagcctt caggtcacgg 20 227 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 227 cacctgcggc tcttcttcgg 20 228 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 228 ttgtctcaca cgagtgcatg 20 229 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 229 ttctgacaat acaattactc 20 230 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 230 ttacagagtc atccacaagc 20 231 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 231 cgatagtctc cacgtgaggc 20 232 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 232 gtaacgctcc tatggccagg 20 233 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 233 agactgactg ctcgtcgcga 20 

What is claimed is:
 1. An antisense compound targeted to a nucleic acid molecule encoding a member of the human Rho family of small GTP binding proteins, wherein said antisense compound inhibits the expression of said member of the human Rho family.
 2. The antisense compound of claim 1 which is an antisense oligonucleotide.
 3. The antisense compound of claim 2 wherein the oligonucleotide comprises at least one modified internucleoside linkage.
 4. The antisense compound of claim 3 wherein the modified internucleoside linkage is a phosphorothioate linkage.
 5. The antisense compound of claim 2 wherein the oligonucleotide comprises at least one modified sugar moiety.
 6. The antisense compound of claim 5 wherein the modified sugar moiety is a 2′-O-methoxyethyl sugar moiety.
 7. The antisense compound of claim 2 wherein the oligonucleotide comprises at least one modified nucleobase.
 8. The antisense compound of claim 7 wherein the modified nucleobase is a 5-methylcytosine.
 9. The antisense compound of claim 2 wherein the oligonucleotide is a chimeric oligonucleotide.
 10. The antisense compound of claim 1 which is from 8 to 30 nucleobases in length.
 11. The antisense compound of claim 1 wherein said member of the human Rho family of small GTP binding proteins is RhoA, RhoB, RhoC, or RhoG.
 12. The antisense compound of claim 1 wherein said member of the human Rho family of small GTP binding protein is rac1.
 13. The antisense compound of claim 12 wherein the oligonucleotide comprises SEQ ID NO: 193, 195, 199, 201, 204 or
 205. 14. The antisense compound of claim 1 wherein said member of the human Rho family of small GTP binding protein is cdc42.
 15. The antisense compound of claim 14 wherein the oligonucleotide comprises SEQ ID NO: 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229 or
 230. 16. A pharmaceutical composition comprising the antisense compound of claim 1 and a pharmaceutically acceptable carrier or diluent.
 17. The pharmaceutical composition of claim 16 further comprising a colloidal dispersion system.
 18. The pharmaceutical composition of claim 16 wherein the antisense compound is an antisense oligonucleotide.
 19. A method of inhibiting the expression of a member of the human Rho family of small GTP binding proteins in human cells or tissues comprising contacting said cells or tissues with the antisense compound of claim 1 so that expression of said human Rho family member is inhibited.
 20. A method of treating a human having a disease or condition associated with a member of the human Rho family of small GTP binding proteins comprising administering to said animal a therapeutically or prophylactically effective amount of the antisense compound of claim 1 so that expression of said human Rho family member is inhibited.
 21. The method of claim 20 wherein the disease or condition is a hyperproliferative condition.
 22. The method of claim 21 wherein the hyperproliferative condition is cancer.
 23. The method of claim 20 wherein the disease or condition is abnormal wound healing or clotting.
 24. The method of claim 20 wherein the disease or condition is ischemia/reperfusion or reoxygenation injury.
 25. A compound which inhibits JNK activation by a non-cytokine activator but does not inhibit JNK activation by a cytokine.
 26. The compound of claim 25 wherein the non-cytokine activator is a stress signal.
 27. The compound of claim 26 wherein the stress signal is hydrogen peroxide or ultraviolet radiation.
 28. The compound of claim 25 wherein the cytokine is IL-1β.
 29. The compound of claim 25 which is an inhibitor of rhoA.
 30. The compound of claim 29 which is an antisense inhibitor of rhoA.
 31. A method of inhibiting JNK activation by a non-cytokine activator without inhibiting JNK activation by a cytokine comprising contacting JNK or cells or tissues containing JNK with an inhibitor of rhoA.
 32. The method of claim 31 wherein the non-cytokine activator is a stress signal.
 33. The method of claim 32 wherein the stress signal is hydrogen peroxide or ultraviolet radiation.
 34. The method of claim 31 wherein the cytokine is IL-1β.
 35. The method of claim 31 wherein the Inhibitor of rhoA is an antisense oligonucleotide inhibitor of rhoA. 